Acridone derivatives as labels for fluorescence detection of target materials

ABSTRACT

Disclosed are methods for assay of an analyte employing acridone dyes having the structure: 
     
       
         
         
             
             
         
       
     
     Z 1  and Z 2  represent atoms necessary to complete one ring, two fused ring, three fused ring aromatic or heteroaromatic systems, each ring having five or six atoms selected from carbon atoms and optionally no more than two atoms selected from oxygen, nitrogen and sulphur; R 2 , R 3 , R 4  and R 5  are selected from hydrogen, halogen, amide, hydroxyl, cyano, nitro, mono- or di-nitro-substituted benzyl, amino, mono- or di-C 1 -C 4  alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C 1 -C 6  alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C 1 -C 20  alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammonium, the groups -E-F and —(CH 2 —) n Y; R 1  is selected from hydrogen, mono- or di-nitro-substituted benzyl, C 1 -C 20  alkyl, aralkyl, the groups -E-F and —(CH 2 —) n Y; where E is a spacer group, F is a target bonding group; Y is selected from sulphonate, sulphate, phosphonate, phosphate, quaternary ammonium and carboxyl.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/943,628 filed Nov. 21, 2007, which is a continuation of U.S. patentapplication Ser. No. 10/479,578 filed Dec. 1, 2003, abandoned, which isa filing under 35 U.S.C. §371 and claims priority to internationalpatent application number PCT/GB2002/002509 filed May 30, 2002,published on Dec. 12, 2002, as WO 2002/099424, which claims priority topatent application number GB 0113435.2 filed Jun. 4, 2001.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to new acridone derivatives havingcharacteristic fluorescence lifetimes that can be used as labels forattachment to and labelling of target materials. The acridonederivatives of the invention may be easily distinguished, one from theother, by virtue of their fluorescence lifetimes and they may be used inmultiparameter applications. The invention also relates to assay methodsutilising acridone derivatives and to a set of different fluorescentacridone lifetime dyes.

2. Description of the Prior Art

There is an increasing interest in, and demand for, fluorescent labelsfor use in the labelling and detection of biological materials.Fluorescent labels are generally stable, sensitive and a wide range ofmethods are now available for the labelling of biomolecules. Typically,the emission spectrum of a fluorescent dye is a characteristic propertyof the dye, the intensity of such emission being used in the detectionof materials labelled with that dye. One problem with measurements offluorescence intensity as a means of detecting and/or measuring theconcentration of a fluorescent labelled biomolecule is that backgroundfluorescence may interfere with the measurement. Thus, in order toobtain improvements in the sensitivity of fluorescence detection, it ishighly desirable to improve the signal-to-noise ratio.

One means of overcoming the problem of background noise has been throughthe use of long wavelength dyes, for example, the cyanine dyes Cy™5 andCy7, as disclosed in U.S. Pat. No. 5,268,486 (Waggoner et al). Thesedyes emit in the 600-800 nm region of the spectrum, where backgroundfluorescence is much less of a problem. Another means of improving thesignal-to-noise ratio in fluorescence measurements is in the use oftime-resolved fluorescence, for example by using fluorescent labelsbased on lanthanide chelates, eg. Eu³⁺ and Tb³⁺ (Selvin et al, U.S. Pat.No. 5,622,821). In time-resolved fluorescent labels, the lifetime of thefluorescence emission is typically longer than that of the backgroundfluorescence, which may therefore be gated out using appropriateinstrumentation.

McGown, L. B. et al (Anal. Chem., (2000), 72, 5865-73) describe the useof a range of different dyes for multiparameter analysis in whichfluorescence lifetime, rather than fluorescence wavelength, is thediscriminating characteristic. Dyes from different dye classes were usedto obtain lifetime resolution; however compensation was required foreither mobility differences or different fluorescence signalintensities. The method has been refined by Sauer, M. et al (J.Fluorescence, (1993), 3 (3), 131-139) who employed a series ofrhodamine-based fluors having a range of fluorescent lifetimes but whichall absorb and emit at similar wavelengths, thus avoiding having tochange the excitation source and emission filters.

The acridone chromophore is highly fluorescent and has been used forlabelling biological molecules and subsequent detection by conventionalfluorescence emission spectroscopy. For example, Faller, T. et al (J.Chem. Soc. Chem. Comm., (1997), 1529-30) describe the preparation of asuccinimidyl ester derivative of acridone and its use in labellingpeptides for subsequent analysis by mass-spectroscopy. U.S. Pat. No.5,472,582 (Jackson) describes the use of the fluorescent label,2-aminoacridone, for labelling and detecting carbohydrates in a mixture,following electrophoretic separation.

Val'kova, G. et al (Dokl. Akad. Nauk. SSR, (1978), 240 (4), 884-7) havemeasured the fluorescence lifetimes of several acridone derivatives,however, to date, there appear to be no reports relating to the use ofacridones as lifetime dyes suitable for labelling and the detection ofbiological materials.

SUMMARY OF THE INVENTION

The present invention therefore describes modifications of the acridonechromophore, to produce a range of acridone derivatives havingcharacteristic fluorescence lifetimes and which are useful for labellingbiological materials.

The acridone derivatives of the present invention moreover provide avaluable set of fluorescent labels having a common core structure andwhich are particularly useful for multiparameter analysis. In each dyeof a set of dyes, the absorption and emission spectra remain essentiallythe same, whilst the fluorescence lifetimes vary. Thus, it is possibleto use a common excitation source and determine the lifetimes at thesame emission wavelength, thereby simplifying requirements for detectioninstrumentation used in multiparameter experiments. Another advantage ofthe present invention is that the fluorescence lifetimes of the acridonedye derivatives are generally longer than the lifetimes of otherfluorescent labels, as well as naturally occurring fluorescentmaterials, such as proteins and polynucleotides, thereby allowing easydiscrimination from background fluorescence in biological assaysutilising such dyes.

Accordingly, in a first aspect of the present invention there isprovided use of a reagent for labelling and lifetime detection of atarget material, wherein said reagent is a dye of the formula (I):

wherein:groups R² and R³ are attached to the Z¹ ring structure and groups R⁴ andR⁵ are attached to the Z² ring structure;Z¹ and Z² independently represent the atoms necessary to complete onering, two fused ring, or three fused ring aromatic or heteroaromaticsystems, each ring having five or six atoms selected from carbon atomsand optionally no more than two atoms selected from oxygen, nitrogen andsulphur;R², R³, R⁴ and R⁵ are independently selected from hydrogen, halogen,amide, hydroxyl, cyano, nitro, mono- or di-nitro-substituted benzyl,amino, mono- or di-C₁-C₄ alkyl-substituted amino, sulphydryl, carbonyl,carboxyl, C₁-C₆ alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl,C₁-C₂₀ alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammonium,the group -E-F and the group —(CH₂—)_(n)Y;R¹ is selected from hydrogen, mono- or di-nitro-substituted benzyl,C₁-C₂₀ alkyl, aralkyl, the group -E-F and the group —(CH₂—)_(n)Y;E is a spacer group having a chain from 1-60 atoms selected from thegroup consisting of carbon, nitrogen, oxygen, sulphur and phosphorusatoms and F is a target bonding group;Y is selected from sulphonate, sulphate, phosphonate, phosphate,quaternary ammonium and carboxyl; and n is an integer from 1 to 6.

In a first embodiment of the first aspect, the dye of formula (I) is afluorescent dye wherein:

groups R² and R³ are attached to the Z¹ ring structure and groups R⁴ andR⁵ are attached to the Z² ring structure, where Z¹ and Z² arehereinbefore defined;R², R³, R⁴ and R⁵ are independently selected from hydrogen, halogen,amide, hydroxyl, cyano, amino, mono- or di-C₁-C₄ alkyl-substitutedamino, sulphydryl, carbonyl, carboxyl, C₁-C₆ alkoxy, acrylate, vinyl,styryl, aryl, heteroaryl, C₁-C₂₀ alkyl, aralkyl, sulphonate, sulphonicacid, quaternary ammonium, the group -E-F and the group —(CH₂—)_(n)Y;andR¹ is selected from hydrogen, C₁-C₂₀ alkyl, aralkyl, the group -E-F andthe group —(CH₂—)_(n)Y;wherein E, F, Y and n are hereinbefore defined.

The acridone dyes according to the first embodiment of the first aspectare particularly suitable for use as fluorescence lifetime dyes. In thecontext of the present invention, the term lifetime dye is intended tomean a dye having a measurable fluorescence lifetime, defined as theaverage amount of time that the dye remains in its excited statefollowing excitation (Lackowicz, J. R., Principles of FluorescenceSpectroscopy, Kluwer Academic/Plenum Publishers, New York, (1999)).

Preferably, the fluorescent dye has a fluorescence lifetime in the rangefrom 2 to 30 nanoseconds, more preferably from 2 to 20 nanoseconds.

In a second embodiment of the first aspect, the dye of formula (I) is anon-fluorescent or substantially non-fluorescent dye wherein:

groups R¹, R², R³, R⁴, R⁵, Z¹ and Z² are hereinbefore defined;and wherein at least one of groups R¹, R², R³, R⁴ and R⁵ comprises atleast one nitro group.

In this embodiment, suitably, the at least one nitro group may beattached directly to the Z¹ and/or Z² ring structures. In thealternative, a mono- or di-nitro-substituted benzyl group may beattached to the R¹, R², R³, R⁴ or R⁵ positions, which optionally may befurther substituted with one or more nitro groups attached directly tothe Z¹ and/or Z² ring structures.

Preferably, in the first and second embodiments, at least one of groupsR¹, R², R³, R⁴ and R⁵ in the dye of formula (I) is the group -E-F whereE and F are hereinbefore defined.

Suitably, the target bonding group F is a reactive or functional group.A reactive group of a compound according to formula (I) can react undersuitable conditions with a functional group of a target material; afunctional group of a compound according to formula (I) can react undersuitable conditions with a reactive group of the target material suchthat the target material becomes labelled with the compound.

Preferably, when F is a reactive group, it is selected from succinimidylester, sulpho-succinimidyl ester, isothiocyanate, maleimide,haloacetamide, acid halide, vinylsulphone, dichlorotriazine,carbodiimide, hydrazide and phosphoramidite. Preferably, when F is afunctional group, it is selected from hydroxy, amino, sulphydryl,imidazole, carbonyl including aldehyde and ketone, phosphate andthiophosphate. By virtue of these reactive and functional groups thecompounds of formula (I) may be reacted with and covalently bond totarget materials.

Suitably, Z¹ and Z² may be selected independently from the groupconsisting of phenyl, pyridinyl, naphthyl, anthranyl, indenyl,fluorenyl, quinolinyl, indolyl, benzothiophenyl, benzofuranyl andbenzimidazolyl moieties. Additional one, two fused, or three fused ringsystems will be readily apparent to the skilled person. Preferably, Z¹and Z² are selected from the group consisting of phenyl, pyridinyl,naphthyl, quinolinyl and indolyl moieties. Particularly preferred Z¹ andZ² are phenyl and naphthyl moieties.

Preferably, at least one of the groups R¹, R², R³, R⁴ and R⁵ of the dyesof formula (I) is a water solubilising group for conferring ahydrophilic characteristic to the compound. Solubilising groups, forexample, sulphonate, sulphonic acid and quaternary ammonium, may beattached directly to the aromatic ring structures Z¹ and/or Z² of thecompound of formula (I). Alternatively, solubilising groups may beattached by means of a C₁ to C₆ alkyl linker chain to said aromatic ringstructures and may be selected from the group —(CH₂—)_(n)Y where Y isselected from sulphonate, sulphate, phosphonate, phosphate, quaternaryammonium and carboxyl; and n is an integer from 1 to 6. Alternativesolubilising groups may be carbohydrate residues, for example,monosaccharides. Examples of water solubilising constituents includeC₁-C₆ alkyl sulphonates, such as —(CH₂)₃—SO₃ ⁻ and —(CH₂)₄—SO₃ ⁻.However, one or more sulphonate or sulphonic acid groups attacheddirectly to the aromatic ring structures of a dye of formula (I) areparticularly preferred. Water solubility may be advantageous whenlabelling proteins.

Suitable spacer groups E may contain 1-60 chain atoms selected from thegroup consisting of carbon, nitrogen, oxygen, sulphur and phosphorus.

For example the spacer group may be:

-   —(CHR′)_(p)—-   —{(CHR′)_(q)—O—(CHR′)_(r)}_(s)—-   —{(CHR′)_(q)—S—(CHR′)_(r)}_(s)—-   —{(CHR′)_(q)—NR′—(CHR′)_(r)}_(s)—-   —{(CHR′)_(q)—(CH═CH)—(CHR′)_(r)}_(s)—-   —{(CHR′)_(q)—Ar—CHR′)_(r)}_(s)—-   —{(CHR′)_(q)—CO—NR′—(CHR′)_(r)}_(s)—-   —{(CHR′)_(q)—CO—Ar—NR′—(CHR′)_(r)}_(s)—    where R′ is hydrogen, C₁-C₄ alkyl or aryl, which may be optionally    substituted with sulphonate, Ar is phenylene, optionally substituted    with sulphonate, p is 1-20, preferably 1-10, q is 0-10, r is 1-10    and s is 1-5.

Specific examples of reactive groups R¹, R², R³, R⁴ and R⁵ and thegroups with which R¹, R², R³, R⁴ and R⁵ can react are provided inTable 1. In the alternative, groups R¹, R², R³, R⁴ and R⁵ may be thefunctional groups of Table 1 that would react with the reactive groupsof a target material.

TABLE 1 Possible Reactive Substituents and Sites Reactive TherewithReactive Groups Functional Groups succinimidyl esters primary amino,secondary amino isothiocyanates amino groups haloacetamides, maleimidessulphydryl, imidazole, hydroxyl, amine acid halides amino groupsanhydrides primary amino, secondary amino, hydroxyl hydrazides,aldehydes, ketones vinylsulphones amino groups dichlorotriazines aminogroups carbodiimides carboxyl groups phosphoramidites hydroxyl groups

Preferred reactive groups which are especially useful for labellingtarget materials with available amino and hydroxyl functional groupsinclude:

where n is 0 or an integer from 1-10.

Aryl is an aromatic substituent containing one or two fused aromaticrings containing 6 to 10 carbon atoms, for example phenyl or naphthyl,the aryl being optionally and independently substituted by one or moresubstituents, for example halogen, hydroxyl, straight or branched chainalkyl groups containing 1 to 10 carbon atoms, aralkyl and C₁-C₆ alkoxy,for example methoxy, ethoxy, propoxy and n-butoxy.

Heteroaryl is a mono- or bicyclic 5 to 10 membered aromatic ring systemcontaining at least one and no more than 3 heteroatoms which may beselected from N, O, and S and is optionally and independentlysubstituted by one or more substituents, for example halogen, hydroxyl,straight or branched chain alkyl groups containing 1 to 10 carbon atoms,aralkyl and C₁-C₆ alkoxy, for example methoxy, ethoxy, propoxy andn-butoxy.

Aralkyl is a C₁ to C₆ alkyl group substituted by an aryl or heteroarylgroup.

Halogen and halo groups are selected from fluorine, chlorine, bromineand iodine.

Exemplary dyes according to the first embodiment of the first aspect areas follows:

-   i) O—(N-succinimidyl)-6-(9-oxo-9H-acridin-10-yl)hexanoate-   ii) O—(N-succinimidyl)-6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoate-   iii) O—(N-succinimidyl)-6-(9-oxo-9H-acridin-4-carboxamido)hexanoate-   iv)    O—(N-succinimidyl)-6-(2-acetamido-9-oxo-9H-acridin-10-yl)hexanoate.

The dyes of the present invention may be used to label and therebyimpart fluorescent properties to a variety of target biologicalmaterials. Thus, in a second aspect, there is provided a method forlabelling a target biological material, the method comprising:

i) adding to a liquid containing said target biological material a dyeof formula (I):

wherein:groups R² and R³ are attached to the Z¹ ring structure and groups R⁴ andR⁵ are attached to the Z² ring structure, where Z¹ and Z² arehereinbefore defined;R², R³, R⁴ and R⁵ are independently selected from hydrogen, halogen,amide, hydroxyl, cyano, amino, mono- or di-C₁-C₄ alkyl-substitutedamino, sulphydryl, carbonyl, carboxyl, C₁-C₆ alkoxy, acrylate, vinyl,styryl, aryl, heteroaryl, C₁-C₂₀ alkyl, aralkyl, sulphonate, sulphonicacid, quaternary ammonium, the group -E-F and the group —(CH₂—)_(n)Y;R¹ is selected from hydrogen, C₁-C₂₀ alkyl, aralkyl, the group -E-F andthe group —(CH₂—)_(n)Y;where E, F, Y and n are hereinbefore defined; andii) incubating said dye with said target biological material underconditions suitable for labelling said target.

Suitably, the fluorescent dyes of the present invention wherein at leastto one of the groups R¹ to R⁵ contains a charge, for example, quaternaryamino, may be used to bind non-covalently to charged biologicalmolecules such as, for example, DNA and RNA. Alternatively, fluorescentdyes of the present invention wherein at least one of the groups R¹ toR⁵ is an uncharged group, for example, a long chain alkyl or an arylgroup, may be used to bind to is uncharged biological molecules such as,for example, biological lipids, as well as to intact cell membranes,membrane fragments and cells.

In a preferred embodiment, at least one of the groups R¹, R², R³, R⁴ andR⁵ in the dye of formula (I) is the group -E-F where E and F arehereinbefore defined. In this embodiment, the fluorescent dyes may beused to covalently label a target biological material. The targetbonding group may be a reactive group for reacting with a functionalgroup of the target material. Alternatively, the target bonding groupmay be a functional group for reacting with a reactive group on thetarget biological material. The method comprises incubating the targetmaterial with an amount of the dye according to the invention underconditions to form a covalent linkage between the target and the dye.The target may be incubated with an amount of a compound according tothe present invention having at least one of groups R¹, R², R³, R⁴ andR⁵ that includes a reactive or functional group as hereinbefore definedthat can covalently bind with the functional or reactive group of thetarget biological material.

Suitable biological materials include, but are not limited to the groupconsisting of antibody, lipid, protein, peptide, carbohydrate,nucleotides which contain or are derivatized to contain one or more ofan amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate andthiophosphate groups, and oxy or deoxy polynucleic acids which containor are derivatized to contain one or more of an amino, sulphydryl,carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups,microbial materials, drugs, hormones, cells, cell membranes and toxins.

The fluorescent dyes according to the invention having a target bondinggroup in at least one of groups R¹, R², R³, R⁴ and R⁵ may be used in anassay method for determining the presence or the amount of an analyte ina sample. Thus, in a third aspect of the present invention, there isprovided a method for the assay of an analyte in a sample which methodcomprises:

i) contacting the analyte with a specific binding partner for saidanalyte under conditions suitable to cause the binding of at least aportion of said analyte to said specific binding partner to form acomplex and wherein one of said analyte and said specific bindingpartner is labelled with a fluorescent dye of formula (I):

wherein:groups R² and R³ are attached to the Z¹ ring structure and groups R⁴ andR⁵ are attached to the Z² ring structure, where Z¹ and Z² arehereinbefore defined;at least one of groups R¹, R², R³, R⁴ and R⁵ is the group -E-F where Eis a spacer group having a chain from 1-60 atoms selected from the groupconsisting of carbon, nitrogen, oxygen, sulphur and phosphorus atoms andF is a target bonding group;when any of said groups R², R³, R⁴ and R⁵ is not said group -E-F, saidremaining groups R², R³, R⁴ and R⁵ are independently selected fromhydrogen, halogen, amide, hydroxyl, cyano, amino, mono- or di-C₁-C₄alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C₁-C₆ alkoxy,acrylate, vinyl, styryl, aryl, heteroaryl, C₁-C₂₀ alkyl, aralkyl,sulphonate, sulphonic acid, quaternary ammonium and the group—(CH₂—)_(n)Y; and,when group R¹ is not said group -E-F, it is selected from hydrogen,C₁-C₂₀ alkyl, aralkyl and the group —(CH₂—)_(n)Y;wherein Y and n are hereinbefore defined;ii) measuring the emitted fluorescence of the labelled complex; andiii) correlating the emitted fluorescence with the presence or theamount of said analyte in said sample.

Suitably, step ii) may be performed by measurement of the fluorescenceintensity or fluorescence lifetime of the sample, preferably thefluorescence lifetime.

In one embodiment, the assay method is a direct assay for themeasurement of an analyte in a sample. Optionally, a known or putativeinhibitor compound may be included in the assay mix.

In a second, or alternative embodiment, the assay may be a competitiveassay wherein a sample containing an analyte competes with a fluorescenttracer for a limited number of binding sites on a binding partner thatis capable of specifically binding the analyte and the tracer. Suitably,the tracer is a labelled analyte or a labelled analyte analogue, inwhich the label is a fluorescent dye of formula (I). Increasing amounts(or concentrations) of the analyte in the sample will reduce the amountof the fluorescent labelled analyte or fluorescent labelled analyteanalogue that is bound to the specific binding partner. The fluorescencesignal is measured and the concentration of analyte may be obtained byinterpolation from a standard curve.

In a further embodiment, the binding assay may employ a two-step format,wherein a first component (which may be optionally coupled to aninsoluble support) is bound to a second component to form a specificbinding complex, which is bound in turn to a third component. In thisformat, the third component is capable of specifically binding to eitherthe second component, or to the specific binding complex. Either of thesecond or the third component may be labelled with a fluorescent dyeaccording to the present invention. Examples include “sandwich” assays,in which one component of a to specific binding pair, such as a firstantibody, is coated onto a surface, such as the wells of a multiwellplate. Following the binding of an antigen to the first antibody, afluorescent labelled second antibody is added to the assay mix, so as tobind with the antigen-first antibody complex. The fluorescence signal ismeasured and the concentration of antigen may be obtained by isinterpolation from a standard curve.

Examples of analyte-specific binding partner pairs include, but are notrestricted to, antibodies/antigens, lectins/glycoproteins,biotin/streptavidin, hormone/receptor, enzyme/substrate or co-factor,DNA/DNA, DNA/RNA and DNA/binding protein. It is to be understood thatany molecules which possess a specific binding affinity for each othermay be employed, so that the fluorescent dyes of the present inventionmay be used for labelling one component of a specific binding pair,which in turn may be used in the detection of binding to the othercomponent.

The fluorescent dyes according to first embodiment of the first aspectmay be used in applications that include detecting and distinguishingbetween various components in a mixture. Thus, in a fourth aspect, thepresent invention provides a set of two or more different fluorescentdyes according to the invention, each dye of said set of dyes having theformula (I):

wherein:groups R² and R³ are attached to the Z¹ ring structure and groups R⁴ andR⁵ are attached to the Z² ring structure, where Z¹ and Z² arehereinbefore defined;R², R³, R⁴ and R⁵ are independently selected from hydrogen, halogen,amide, hydroxyl, cyano, amino, mono- or di-C₁-C₄ alkyl-substitutedamino, sulphydryl, carbonyl, carboxyl, C₁-C₆ alkoxy, acrylate, vinyl,styryl, aryl, heteroaryl, C₁-C₂₀ alkyl, aralkyl, sulphonate, sulphonicacid, quaternary ammonium, the group -E-F and the group —(CH₂—)_(n)Y;R¹ is selected from hydrogen, C₁-C₂₀ alkyl, aralkyl, the group -E-F andthe group —(CH₂—)_(n)Y;E is a spacer group having a chain from 1-60 atoms selected from thegroup consisting of carbon, nitrogen, oxygen, sulphur and phosphorusatoms and F is a target bonding group;Y is selected from sulphonate, sulphate, phosphonate, phosphate,quaternary ammonium and carboxyl; and n is an integer from 1 to 6;wherein each dye of said set has a distinguishably differentfluorescence lifetime compared with the lifetimes of the remaining dyesof the set.

Preferably, in each dye of the set of dyes at least one of groups R¹,R², R³, R⁴ and R⁵ is the group -E-F where E and F are hereinbeforedefined.

Preferably, the set of fluorescent dyes according to the invention willcomprise four different dyes, each dye of the set having a differentfluorescence lifetime.

Preferably, each of the fluorescent dyes in the set has a fluorescencelifetime in the range from 2 to 30 nanoseconds. More preferably thefluorescent dyes in the set will have fluorescence lifetimes in therange from 2 to 20 nanoseconds.

To distinguish between different dyes in the set of dyes, the differencein the lifetimes of the fluorescent emission of two such dyes ispreferably at least 15% of the value of the shorter lifetime dye.

The set of dyes may be used in a detection method wherein differentfluorescent dyes of the set of dyes are covalently bonded to a pluralityof different primary components, each primary component being specificfor a different secondary component, in order to identify each of aplurality of secondary components in a mixture of secondary components.The method comprises covalently binding different dyes of a set offluorescent dyes according to the fourth aspect of the invention todifferent primary components in a multicomponent mixture wherein eachdye of the set has a different fluorescence lifetime, compared with thefluorescence lifetimes of the remaining dyes of the set; adding thedye-labelled primary components to a preparation containing secondarycomponents under conditions to enable binding of at least a portion ofeach of said dye-labelled primary components to its respective secondarycomponent; and determining the presence or the amount of the boundsecondary component by measuring the fluorescence lifetime of each ofthe labelled primary component-secondary component complexes.

If required, any unreacted primary components may be removed orseparated from the preparation by, for example washing, to preventinterference with the analysis.

Preferably, a single wavelength of excitation can be used to excitefluorescence from two or more materials in a mixture, where eachfluoresces having a different characteristic fluorescent lifetime.

The set of fluorescent dyes according to the present invention may beused in any system in which the creation of a fluorescent primarycomponent is possible. For example, an appropriately reactivefluorescent dye according to the invention can be conjugated to a DNA orRNA fragment and the resultant conjugate then caused to bind to acomplementary target strand of DNA or RNA. Other examples of primarycomponent-secondary component complexes which may be detected includeantibodies/antigens and biotin/streptavidin.

The set of dyes according to the present invention may also beadvantageously used in fluorescent DNA sequencing based uponfluorescence lifetime discrimination of the DNA fragments. Briefly, eachone of a set of dyes, may be coupled to a primer. Various primers areavailable, such as primers from pUC/M13, λgt10, λgt11 and the like (seeSambrook et al, Molecular Cloning, A Laboratory Manual 2^(nd) Edition,Cold Spring Harbour Laboratory Press 1989). DNA sequences are clonedinto an appropriate vector having a primer sequence joined to the DNAfragment to be sequenced. After hybridisation to the DNA template,polymerase enzyme-directed synthesis of a complementary strand occurs.Different 2′,3′-dideoxynucleotide terminators are employed in eachdifferent sequencing reaction so as to obtain base-specific terminationof the chain extension reaction. The resulting set of DNA fragments areseparated by electrophoresis and the terminating nucleotide (and thusthe DNA sequence) is determined by detecting the fluorescence lifetimeof the labelled fragments. DNA sequencing may also be performed usingdideoxynucleotide terminators covalently labelled with the fluorescentdyes according to the present invention.

The non-fluorescent or substantially non-fluorescent dyes according tothe second embodiment of the first aspect may be used as the substratefor an enzyme and which upon reaction with the enzyme, yields afluorescent product.

Bacterial nitroreductases have been shown to catalyse the generalreaction set out below in Reaction Scheme 1.

where, in the presence of NADH or NADPH, one or more nitro groups on anorganic molecule may be reduced to a hydroxylamine (—NHOH) group whichto may subsequently be converted to an amine (—NH₂) group.

Thus, in a fifth aspect of the invention, there is provided a method ofincreasing the fluorescence of a dye of formula (I):

wherein:groups R¹, R², R³, R⁴, R⁵, Z¹ and Z² are hereinbefore defined andwherein at least one of groups R¹, R², R³, R⁴ and R⁵ comprises at leastone nitro group; characterised by the reduction of said at least onenitro group to —NHOH or —NH₂

Preferably, the fluorescence lifetime of the fluorescent product of thereduction is in the range from 2 to 30 nanoseconds.

Suitably, reduction is by means of nitroreductase. This can be achievedby enzymatic conversion of a nitro group in a compound of formula (I) toa —NHOH or —NH₂ group by the action of the nitroreductase. Depending onthe structure of the dye, the fluorescence emission from the product ofthe nitroreductase reaction may typically have a lifetime in the range 2to 30 nanoseconds. Moreover, the fluorescence lifetime characteristicsof the reaction product can be altered to suit the application by meansof additional substitutents, whilst retaining the nitro group(s) thatare involved in the reaction with nitroreductase. Thus, fluorescentreporters compatible for use with other fluors in multiplex systems canbe provided.

In a sixth aspect of the invention there is provided a method fordetecting nitroreductase enzyme activity in a composition comprising:

i) mixing under conditions to promote nitroreductase activity saidcomposition with a dye of formula (I):

wherein:groups R¹, R², R³, R⁴, R⁵, Z¹ and Z² are hereinbefore defined andwherein at least one of groups R¹, R², R³, R⁴ and R⁵ comprises at leastone nitro group; andii) measuring an increase in fluorescence wherein said increase is ameasure of the amount of nitroreductase activity.

Suitably, the measurement of step ii) may be of the fluorescenceintensity and/or fluorescence lifetime of the dye.

In one embodiment of the sixth aspect, the composition comprises a cellor cell extract. In principle, any type of cell can be used, i.e.prokaryotic or eukaryotic (including bacterial, mammalian and plantcells). Where appropriate, a cell extract can be prepared from a cell,using standard methods known to those skilled in the art (MolecularCloning, A Laboratory Manual 2^(nd) Edition, Cold Spring HarbourLaboratory Press 1989), prior to measuring fluorescence.

Typical conditions for nitroreductase activity comprise incubation ofthe composition in a suitable medium and the dye at approximately 37° C.in the presence of NADH and FMN.

In a seventh aspect of the invention there is provided an assay methodcomprising:

i) binding one component of a specific binding pair to a surface;ii) adding a second component of the specific binding pair underconditions to promote binding between the components, said secondcomponent being labelled with a nitroreductase enzyme;iii) adding a dye of formula (I):

wherein:groups R¹, R², R³, R⁴, R⁵, Z¹ and Z² are hereinbefore defined andwherein at least one of groups R¹, R², R³, R⁴ and R⁵ comprises at leastone nitro group; andiv) detecting binding of the second component to the first component bymeasuring an increase in fluorescence as a measure of boundnitroreductase activity.

In a preferred embodiment of the seventh aspect, said specific bindingpair is selected from the group consisting of antibodies/antigens,lectins/glycoproteins, biotin/streptavidin, hormone/receptor,enzyme/substrate, DNA/DNA, DNA/RNA and DNA/binding protein.

Briefly, an in vitro assay method for the detection of antibody bindingmay be configured as follows. An antibody specific for an antigen ofinterest may be labelled by covalently linking it to an enzymaticallyactive nitroreductase. The labelled antibody can then be introduced intothe test sample containing the antigen under conditions suitable forbinding. After washing to remove any unbound antibody, the amount ofbound antibody is detected by incubating the sample with a substratecomprising a compound of formula (I) having at least one nitro groupunder conditions for nitroreductase activity and measuring an increasein fluorescence. The amount of fluorescence detected will beproportional to the amount of nitroreductase-labelled antibody that hasbound to the antigen.

In an in vitro assay for detecting the binding of nucleic acids byhybridisation, either of the pair of target and probe nucleic acid isimmobilised by attachment to a membrane or surface. The second member ofthe pair is labelled with nitroreductase and incubated under hybridisingconditions with the immobilised nucleic acid. Unbound, labelled nucleicacid is washed off and the amount of bound, labelled nucleic acid ismeasured by incubating the membrane or surface with a compound offormula (I) having at least one nitro group under conditions suitablefor nitroreductase activity. The amount of increase in fluorescencegives a measure of the amount of bound labelled DNA.

Methods for coupling enzymes, such as nitroreductase, to otherbiomolecules, e.g. proteins and nucleic acids, are well known(Bioconjugate Techniques, Academic Press 1996). Coupling may be achievedby direct means, for example by use of a suitable bifunctionalcrosslinking agent (e.g. N-[γ-maleimidopropionic acid]hydrazine, Pierce)to covalently link the enzyme and binding partner. Alternatively,coupling may be achieved by indirect means, for example by separatelybiotinylating the enzyme and the binding partner using a chemicallyreactive biotin derivative, (e.g. N-hydroxysuccinimido-biotin, Pierce)and subsequently coupling the molecules through a streptavidin bridgingmolecule.

Cell based assays are increasingly attractive over in vitro biochemicalassays for use in high throughput screening (HTS). This is because cellbased assays require minimal manipulation and the readouts can beexamined in a biological context that more faithfully mimics the normalphysiological situation. Such in vivo assays require an ability tomeasure a to cellular process and a means to measure its output. Forexample, a change in the pattern of transcription of a number of genescan be induced by cellular signals triggered, for example, by theinteraction of an agonist with its cell surface receptor or by internalcellular events such as DNA damage. The induced changes in transcriptioncan be identified by fusing a reporter gene to is a promoter regionwhich is known to be responsive to the specific activation signal.

In fluorescence-based enzyme-substrate systems, an increase influorescence gives a measure of the activation of the expression of thereporter gene.

Accordingly, in a eighth aspect of the invention, there is provided anassay method which comprises:

i) contacting a host cell which has been transfected with a nucleic acidmolecule comprising expression control sequences operably linked to asequence encoding a nitroreductase, with a dye of formula (I):

wherein:groups R¹, R², R³, R⁴, R⁵, Z¹ and Z² are hereinbefore defined andwherein at least one of groups R¹, R², R³, R⁴ and R⁵ comprises at leastone nitro group; andii) measuring an increase in fluorescence as a measure of nitroreductasegene expression.

In one embodiment of the eighth aspect, the assay method is conducted inthe presence of a test agent whose effect on gene expression is to bedetermined.

Methods for using a variety of enzyme genes as reporter genes inmammalian cells are well known (for review see Naylor L. H., BiochemicalPharmacology, (1999), 58, 749-757). The reporter gene is chosen to allowis the product of the gene to be measurable in the presence of othercellular proteins and is introduced into the cell under the control of achosen regulatory sequence which is responsive to changes in geneexpression in the host cell. Typical regulatory sequences include thoseresponsive to hormones, second messengers and other cellular control andsignalling factors. For example, agonist binding to seven transmembranereceptors is known to modulate promoter elements including the cAMPresponsive element, NF-AT, SRE and AP1; MAP kinase activation leads tomodulation of SRE leading to Fos and Jun transcription; DNA damage leadsto activation of transcription of DNA repair enzymes and the tumoursuppressor gene p53. By selection of an appropriate regulatory sequencethe reporter gene can be used to assay the effect of added agents oncellular processes involving the chosen regulatory sequence under study.

For use as a reporter gene, the nitroreductase gene may be isolated bywell known methods, for example by amplification from a cDNA library byuse of the polymerase chain reaction (PCR) (Molecular Cloning, ALaboratory Manual 2^(nd) Edition, Cold Spring Harbour Laboratory Press(1989) pp 14.5-14.20). Once isolated, the nitroreductase gene may beinserted into a vector suitable for use with mammalian promoters(Molecular Cloning, A Laboratory Manual 2^(nd) Edition, Cold SpringHarbour Laboratory Press (1989) pp 16.56-16.57) in conjunction with andunder the control of the gene regulatory sequence under study. Thevector containing the nitroreductase reporter and associated regulatorysequences may then be introduced into the host cell by transfectionusing well known techniques, for example by use of DEAE-Dextran orCalcium Phosphate (Molecular Cloning, A Laboratory Manual 2^(nd)Edition, Cold Spring Harbour Laboratory Press (1989) pp 16.30-16.46).Other suitable techniques will be well known to those skilled in theart.

In another embodiment of the eighth aspect, the dye of formula (I)wherein groups R¹, R², R³, R⁴, R⁵, Z¹ and Z² are hereinbefore definedand wherein at least one of groups R¹, R², R³, R⁴ and R⁵ comprises atleast one nitro group, is permeable to cells. In this embodiment,preferably, at least one is of groups R¹, R², R³, R⁴ or R⁵ comprises acell membrane permeabilising group. Membrane permeant compounds can begenerated by masking hydrophilic groups to provide more hydrophobiccompounds. The masking groups can be designed to be cleaved from thesubstrate within the cell to generate the derived substrateintracellularly. Because the substrate is more hydrophilic than themembrane permeant derivative it is then trapped in the cell. Suitablecell membrane permeabilising groups may be selected from acetoxymethylester which is readily cleaved by endogenous mammalian intracellularesterases (Jansen, A. B. A. and Russell, T. J., J. Chem. Soc., (1965),2127-2132 and Daehne W. et al. J. Med. Chem., (1970) 13, 697-612) andpivaloyl ester (Madhu et al., J. Ocul. Pharmacol. Ther., (1998), 14 (5),389-399) although other suitable groups will be recognised by thoseskilled in the art.

Typically, to assay the activity of a test agent to activate cellularresponses via the regulatory sequence under study, cells transfectedwith the nitroreductase reporter are incubated with the test agent,followed by addition of a dye of formula (I) wherein at least one ofgroups R¹, R², R³, R⁴ and R⁵ in said dye comprises at least one nitrogroup, said compound being made cell permeant. After an appropriateperiod required for conversion of the substrate to a form exhibitingfluorescence, the fluorescence from the cells is measured at an emissionwavelength appropriate for the chosen dye. Measurement of fluorescencemay be readily achieved by use of a range of detection instrumentsincluding fluorescence microscopes (e.g. LSM 410, Zeiss), microplatereaders (e.g. CytoFluor 4000, Perkin Elmer), CCD imaging systems (e.g.LEADseeker™, Amersham Pharmacia Biotech) and Flow Cytometers (e.g.FACScalibur, Becton Dickinson).

The measured fluorescence is compared with fluorescence from controlcells not exposed to the test agent and the effects, if any, of the testagent on gene expression modulated through the regulatory sequence, isdetermined by the detection of the characteristic fluorescence in thetest cells. Where appropriate, a cell extract can be prepared usingconventional is methods.

Suitable means for expressing a nitroreductase enzyme include anexpression plasmid or other expression construct. Methods for preparingsuch expression constructs are well known to those skilled in the art.

In an ninth aspect of the present invention, there is provided a dye offormula (I):

wherein:groups R² and R³ are attached to the Z¹ ring structure and groups R⁴ andR⁵ are attached to the Z² ring structure;Z¹ and Z² independently represent the atoms necessary to complete onering, two fused ring, or three fused ring aromatic or heteroaromaticsystems, each ring having five or six atoms selected from carbon atomsand optionally no more than two atoms selected from oxygen, nitrogen andsulphur; at least one of groups R¹, R², R³, R⁴ and R⁵ is the group -E-Fwhere E is a spacer group having a chain from 1-60 atoms selected fromthe group consisting of carbon, nitrogen, oxygen, sulphur and phosphorusatoms and F is a target bonding group; and,when any of said groups R¹, R², R³, R⁴ and R⁵ is not said group -E-F,said remaining groups R², R³, R⁴ and R⁵ are independently selected fromhydrogen, halogen, amide, hydroxyl, cyano, nitro, amino, mono- ordi-C₁-C₄ alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C₁-C₆alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C₁-C₂₀ alkyl,aralkyl, sulphonate, sulphonic acid, quaternary ammonium and the group—(CH₂—)_(n)Y; and,when group R¹ is not said group -E-F, it is selected from hydrogen,mono- or di-nitro-substituted benzyl, C₁-C₂₀ alkyl, aralkyl and thegroup —(CH₂—)_(n)Y;E is a spacer group having a chain from 1-60 atoms selected from thegroup consisting of carbon, nitrogen, oxygen, sulphur and phosphorusatoms and F is a target bonding group;Y is selected from sulphonate, sulphate, phosphonate, phosphate,quaternary ammonium and carboxyl; and n is an integer from 1 to 6;provided that at least one of groups R¹, R², R³, R⁴ and R⁵ is a watersolubilising group.

Preferably, the target bonding group F comprises a reactive group forreacting with a functional group on a target material, or a functionalgroup for reacting with a reactive group on a target material. Preferredreactive groups may be selected from carboxyl, succinimidyl ester,sulpho-succinimidyl ester, isothiocyanate, maleimide, haloacetamide,acid halide, hydrazide, vinylsulphone, dichlorotriazine andphosphoramidite. Preferred functional groups may be selected fromhydroxy, amino, sulphydryl, imidazole, carbonyl including aldehyde andketone, phosphate and thiophosphate.

Preferably, the spacer group E is selected from:

-   —(CHR′)_(p)—-   —{(CHR′)_(q)—O—(CHR′)_(r)}_(s)—-   —{(CHR′)_(q)—NR′—(CHR′)_(r)}_(s)—-   —{(CHR′)_(q)—(CH═CH)—(CHR′)_(r)}_(s)—-   —{(CHR′)_(q)—Ar—(CHR)_(r)}_(s)—-   —{(CHR′)_(q)—CO—NR′—(CHR′)_(r)}_(s)—-   —{(CHR′)_(q)—CO—Ar—NR′—(CHR′)_(r)}_(s)—    where R′ is hydrogen, C₁-C₄ alkyl or aryl, which may be optionally    substituted with sulphonate, Ar is phenylene, optionally substituted    with sulphonate, p is 1-20, preferably 1-10, q is 0-10, r is 1-10    and s is 1-5.

The dyes of formula (I) may be prepared from the correspondingdiphenylamine-2-carboxylic acid according to published methods (seeAlbert, A. and Ritchie, B., Org. Syntheses, (1942), 22, 5; also U.S.Pat. No. 3,021,334). Suitably, the diphenylamine-2-carboxylic acid maybe heated in the presence of an acidic dehydrating agent such asphosphorus oxychloride or concentrated sulfuric acid. Thediphenylamine-2-carboxylic acid derivatives may be prepared by reactionof a 2-halobenzoic acid with a suitable primary aminobenzene (having atleast one aryl ring position unsubstituted ortho- to the amino group),which reaction may be performed in the presence of catalytic coppermetal/salt (see Ullmann, F., Chem. Ber., (1903), 36, 2382; also BritishPatent 649147). Suitably, the 2-halobenzoic acid is heated with theaminobenzene, in the presence of a base such as an alkali metalcarbonate, in a solvent such as 1-butanol or 1-pentanol. A catalyticamount of copper metal powder or a copper salt such as anhydrous copperacetate is also usually included, although sometimes this is notrequired.

It will be readily appreciated that certain dyes of the presentinvention may be useful as intermediates for conversion to other dyes bymethods well known to those skilled in the art. The dyes of the presentinvention may be synthesized by the methods disclosed herein.Derivatives of the compounds having a particular utility are preparedeither by selecting appropriate precursors or by modifying the resultantcompounds by known methods to include functional groups at a variety ofpositions. As examples, the dyes of the present invention may bemodified to include certain reactive groups for preparing a dyeaccording to the present invention, or charged or polar groups may beadded to enhance the solubility of the compound in polar or nonpolarsolvents or materials. As examples of conversions, an ester may beconverted to a carboxylic acid or may be converted to an amidoderivative. Groups R¹ to R⁵ may be chosen so that the dyes of thepresent invention have different lifetime characteristics, therebyproviding a number of related dyes which can be used in multiparameteranalyses wherein the presence and quantity of different compounds in asingle sample may be differentiated based on the wavelengths andlifetimes of a number of detected fluorescence emissions. The dyes ofthe present invention may be made soluble in aqueous, other polar, ornon-polar media containing the material to be labelled by appropriateselection of R-groups.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the absorbance spectra (1A) and the emission spectra (1B)of four acridone dyes according to the present invention;

FIG. 2 shows the fluorescence lifetime decay plot of four acridone dyesaccording to the present invention, as follows:

--------------: N-(succinyl)-2-amino-10H-acridine-9-one;

     : 6-(9-oxo-9H-acridin-10-yl)hexanoic acid;

--- --- --- ---: 6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoic acid;

  ---   ---: 6-(9-oxo-9H-acridin-4-carboxamido) hexanoic acid;

FIG. 3 shows lifetime decay plots of protein conjugates as follows:Conjugate 1=6-(9-oxo-9H-acridin-10-yl)hexanoic acid—bovine serum albumin(BSA) conjugate; Conjugate 2=6-(9-oxo-9H-acridin-4-carboxamido) hexanoicacid—rabbit serum albumin conjugate;

FIG. 4 is a lifetime decay plot following immunoprecipitation withanti-BSA antibody as described in Example 16;

FIG. 5 illustrates fluorescence lifetime detection in capillaryelectrophoresis of four acridone dye-labelled DNA fragments as describedin Example 17;

FIG. 6 shows the lifetime detection of a mixture of two differentacridone dye-labelled BSA conjugates and co-electrophoresed in SDS PAGEas described in Example 19.

Cy™ is a trademark of Amersham Biosciences UK Limited.

EXAMPLES

The present examples are provided for illustrative purposes only, andare not to be construed as limiting the present invention as defined bythe appended claims. All references given below and elsewhere in thepresent specification are hereby included herein by reference.

1. O—(N-Succinimidyl)-6-(9-oxo-9H-acridin-10-yl)hexanoate

1.1 O-Ethyl-6-(9-oxo-9H-acridin-10-yl)hexanoate

9-(10H)-Acridone (4.88 g, 25 mmol) was mixed with anhydrous methylsulfoxide (25 ml) under nitrogen atmosphere and set stirring. After 5minutes, the resultant yellow slurry was treated with potassiumtert-butoxide (3.37 g, 30 mmol) and stirring continued until all thesolids had dissolved. Ethyl 6-bromohexanoate (6.7 g, 30 mmol) was thenadded and the resulting solution stirred under nitrogen for 3 days. Atthe end of this time the reaction mixture was poured into water (1000ml) and extracted with ethyl acetate. The organic solution was washedwith 0.5M aqueous HCl, then with water, before being dried (MgSO₄),filtered and evaporated under vacuum.

The crude product was separated from unreacted acridone by triturationwith 1:1 ethyl acetate/hexane; acridone remained undissolved and wasfiltered off. The filtrate was washed twice with 0.5M aqueous HCl beforebeing dried (MgSO₄), filtered and evaporated under vacuum; the acid washremoved most of the O-alkylated acridine side product. The residue wasthen subjected to flash column chromatography (silica. 30-50% ethylacetate/hexane) to give 5.9 g (70%) ofO-ethyl-6-(9-oxo-9H-acridin-10-yl)hexanoate. This material was finallyrecrystallized from ethanol (20 ml) to give 5.14 g of analytically purematerial. λ_(max) (EtOH)=404, 387, 254 nm; δ_(H) (300 MHz, CD₃OD) 1.23(3H, t), 1.60 (2H, m), 1.73 (2H, m), 1.92 (2H, m), 2.37 (2H, t), 4.11(2H, q), 4.50 (2H, app.t) 7.34 (2H, m), 7.77 (2H, m), 7.84 (2H, m) and8.46 (2H, m). Mass spectrum: (ES+) 338 (M+H), 360 (M+Na).

1.2 6-(9-Oxo-9H-acridin-10-yl)hexanoic acid

O-Ethyl-6-(9-oxo-9H-acridin-10-yl)hexanoate (3.40 g, 10 mmol) was mixedwith acetic acid (40 ml) and 1.0M aqueous HCl (10 ml). The resultingsolution was heated under reflux for 3 hrs until TLC indicated completeconversion to the carboxylic acid (RPC₁₈. Methanol, 90: water, 10.R_(f)=0.6). The solution was evaporated under vacuum, then co-evaporatedwith acetonitrile until a yellow solid was obtained. This was trituratedwith diethyl ether and dried under high vacuum over phosphorus pentoxideto give 6-(9-oxo-9H-acridin-10-yl)hexanoic acid (3.07 g, 98%). λ_(max)(EtOH)=404, 384, 256 nm. δ_(H) (300 MHz, CD₃OD) 1.62 (2H, m), 1.75 (2H,m), 1.95 (2H, m), 2.36 (2H, t), 4.53 (2H, app.t), 7.35 (2H, m), 7.79(2H, m), 7.86 (2H, m) and 8.46 (2H, m). Mass spectrum: (ES+) 310 (M+H),332 (M+Na). Melting Point=167° C.

1.3 O—(N-Succinimidyl)-6-(9-oxo-9H-acridin-10-yl)hexanoate

6-(9-Oxo-9H-acridin-10-yl)hexanoic acid (309 mg, 1.0 mmol) andO—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TSTU;301 mg, 1.0 mmol) were dissolved in N,N-dimethylformamide (5 ml). To theresulting solution was added N,N-diisopropylethylamine (183 μl, 1.05mmol). After 2 hrs the solvent was evaporated under vacuum. The residuewas purified by flash chromatography (silica. 0-10% ethylacetate/dichloromethane) to giveO—(N-succinimidyl)-6-(9-oxo-9H-acridin-10-yl)hexanoate as a pale yellowpowder (330 mg, 81%). δ_(H) (200 MHz, DMSO-d₆) 1.63-1.83 (6H, m), 2.74(2H, t), 2.83 (4H, s), 4.47 (2H, app.t), 7.30-7.38 (2H, m), 7.81-7.84(4H, m) and 8.34-8.38 (2H, m). Mass spectrum: (ES+) 407 (M+H), 429(M+Na). Accurate mass: (M+H)=C₂₃H₂₃N₂O₅, requires 407.1607. Found407.1597 (−2.4 ppm).

2. O—(N-Succinimidyl)-6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoate

2.1 O-Ethyl-6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoate andO-ethyl-6-(2,7-dibromo-9-oxo-9H-acridin-10-yl)hexanoate

O-Ethyl-6-(9-oxo-9H-acridin-10-yl)hexanoate (4.22 g, 12.5 mmol) wasdissolved in ethanol (150 ml) with stirring. To the resulting solutionwas added benzyltrimethylammoniun tribromide (9.75 g, 25 mmol). Themixture was stirred under nitrogen for 4 days. The solvent was thenevaporated under vacuum and the residue partitioned between water (1000ml) and ethyl acetate (300 ml). The organic layer was collected, washedwith more water and dilute aqueous sodium thiosulfate solution, thendried (MgSO₄), filtered and evaporated under vacuum.

The crude product was purified by flash column chromatography (silica.0-5% ethyl acetate/dichloromethane).O-Ethyl-6-(2,7-dibromo-9-oxo-9H-acridin-10-yl)hexanoate eluted first,followed by O-ethyl-6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoate. Purefractions of each were pooled and evaporated separately.

O-Ethyl-6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoate was recrystallizedfrom ethanol. Yield: 2.77 g (53%). λ_(max) (EtOH)=412, 392, 300, 278,256 nm. δ_(H) (300 MHz, CD₃OD) 1.22 (3H, t), 1.58 (2H, m), 1.74 (2H, m),1.91 (2H, m), 2.37 (2H, t), 4.10 (2H, q), 4.49 (2H, app.t), 7.36 (1H,m), 7.72 (1H, d), 7.78 (1H, d), 7.82-7.92 (2H, m), 8.42 (1H, dd) and8.52 (1H, dd). Mass spectrum: (ES+) 416 and 418 (M+H), 438 and 440(M+Na).

O-Ethyl-6-(2,7-dibromo-9-oxo-9H-acridin-10-yl)hexanoate wasrecrystallized from chloroform/ethanol. Yield: 2.06 g (33%). δ_(H) (200MHz, DMSO-d₆) 1.15 (3H, t), 1.50-1.80 (6H, m), 2.31 (2H, t), 4.04 (2H,q), 4.43 (2H, app.t), 7.81 (2H, d), 7.95 (2H, dd) and 8.34 (2H, d).

2.2 6-(2-Bromo-9-oxo-9H-acridin-10-yl)hexanoic acid

O-Ethyl-6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoate (2.5 g, 6 mmol) wasdissolved in acetic acid (30 ml). To this solution was added 1.0Maqueous HCl (10 ml). The mixture was heated under reflux for 3.5 hrs.Reverse phase chromatographic analysis (C₁₈) (methanol:water, 90:10)indicated two spots at R_(f)=0.3 and R_(f)=0.55. The solution was thenevaporated under vacuum, then co-evaporated with acetonitrile until ayellow solid was obtained. This was triturated with diethyl ether anddried under high vacuum over phosphorus pentoxide to give6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoic acid (2.27 g, 97%). δ_(H)(200 MHz, DMSO-d₆) 1.50-1.65 (4H, m), 1.70-1.81 (2H, m), 2.25 (2H, t),4.46 (2H, app.t), 7.32-7.40 (1H, m), 7.78-7.98 (4H, m) and 8.31-8.41(2H, m). λ_(max) (EtOH)=414, 397, 256 nm. Melting Point=213° C.

2.3 O—(N-Succinimidyl)-6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoate

6-(2-Bromo-9-oxo-9H-acridin-10-yl)hexanoic acid (388 mg, 1.0 mmol) andO—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TSTU;301 mg, 1.0 mmol) were dissolved in N,N-dimethylformamide (5 ml). To theresulting solution was added N,N-diisopropylethylamine (183 μl, 1.05mmol). After 2 hrs the solvent was evaporated under vacuum. The residuewas purified by flash chromatography (silica. 0-10% ethylacetate/dichloromethane) to giveO—(N-succinimidyl)-6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoate (330 mg,87%) δ_(H) (200 MHz, DMSO-d₆) 1.5-1.8 (6H, m), 2.73 (2H, t), 2.80 (4H,s), 4.47 (2H, app.t), 7.32-7.42 (1H, m), 7.78-7.83 (3H, m), 7.94 (1H,dd), 8.35 (1H, d) and 8.41 (1H, d). Mass spectrum: (ES+) 485+487 (M+H),507/509 (M+Na). Accurate mass: (M+H)=C₂₃H₂₂BrN₂O₅, requires 485.0712.Found 485.0689 (−4.8 ppm).

3. O—(N-Succinimidyl)-6-(2,7-dibromo-9-oxo-9H-acridin-10-yl)hexanoate

3.1 6-(2,7-Dibromo-9-oxo-9H-acridin-10-yl)hexanoic acid

O-Ethyl-6-(2,7-dibromo-9-oxo-9H-acridin-10-yl)hexanoate (2.0 g, 4.04mmol) was dissolved in acetic acid (30 ml). To this solution was added1.0M aqueous HCl (10 ml). The mixture was heated under reflux for 4 hrs.Reverse phase chromatographic analysis (C₁₈) (methanol:water, 90:10)indicated two spots at R_(f)=0.2 and R_(f)=0.4. The solution was allowedto cool to ambient temperature, whereupon the product crystallized asfluffy yellow needles. After final cooling in an ice bath, the solid wascollected by vacuum filtration, washed with cold aqueous acetic acid,then diethyl ether and dried under vacuum over phosphorus pentoxide togive 6-(2,7-dibromo-9-oxo-9H-acridin-10-yl)hexanoic acid (1.80 g, 97%).δ_(H) (200 MHz, DMSO-d₆) 1.50-1.80 (6H, m), 2.25 (2H, t), 4.40 (2H,app.t), 7.78 (2H, d), 7.92 (2H, dd) and 8.3 (2H, d).

3.2 O—(N-Succinimidyl)-6-(2,7-dibromo-9-oxo-9H-acridin-10-yl)hexanoate

6-(2,7-Dibromo-9-oxo-9H-acridin-10-yl)hexanoic acid (467 mg, 1.0 mmol)and O—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TSTU; 301 mg, 1.0 mmol) were dissolved in N,N-dimethylformamide (5 ml).To the resulting solution was added N,N-diisopropylethylamine (183 μl,1.05 mmol). After leaving to stand overnight, the is solvent wasevaporated under vacuum. The residue was purified by flashchromatography (silica. 10% ethyl acetate/dichloromethane) to giveO—(N-succinimidyl)-6-(2,7-dibromo-9-oxo-9H-acridin-10-yl)hexanoate (510mg, 91%). δ_(H) (200 MHz, DMSO-d₆) 1.5-1.8 (6H, m), 2.72 (2H, t), 2.83(4H, s), 4.44 (2H, app.t), 7.82 (2H, d), 7.95 (2H, dd) and 8.36 (2H, d).Mass spectrum: (ES+) 563+565+567 (M+H), 585+587+589 (M+Na). Accuratemass: (M+Na)=C₂₃H₂₀Br₂N₂O₅Na, requires 584.9637. Found 584.9608 (−4.9ppm).

4. O—(N-Succinimidyl)-6-(9-oxo-9H-acridin-4-carboxamido)hexanoate

4.1 4-Carboxyacridone

2,2′-Iminodibenzoic acid (5.27 g, 20.5 mmol) was mixed with phosphorusoxychloride (20 ml). The resulting pale yellow slurry was heated toboiling. The slurry turned initially bright yellow, then dissolved togive a deep red solution which was intensely yellow at the meniscus.After 2 hrs at reflux, excess solvent was evaporated under vacuum togive a dark oil. This was quenched with ice, then diluted with 2.0Maqueous HCl (25 ml) and the resulting dark solution re-heated toboiling. After 20 mins a solid precipitated to and the mixture becamevery thick; another 20 mls of water was then added to allow effectivestirring. After 1.5 hrs, the mixture was allowed to cool to ambienttemperature. The yellow solid was collected by vacuum filtration, washedwell with water, then acetone, and dried under vacuum to give4-carboxyacridone (4.61 g, 94%). λ_(max) (EtOH)=408, 390, 256 nm. δ_(H)(300 MHz, DMSO-d₆) 7.24-7.33 (2H, m), 7.67-7.76 (2H, m), 8.17 (1H, d),8.38 (1H, dd), 8.47 (1H, dd) and 11.9 (broad s, partially exch). Massspectrum: (ES+) 240 (M+H), 262 (M+Na). Melting Point>300° C.

4.2 6-(9-Oxo-9H-acridin-4-carboxamido)hexanoic acid

4-Carboxyacridone (2.15 g, 9 mmol), was mixed with N,N-dimethylformamide(15 ml) and N,N-diisopropylethylamine (1.6 ml, 9.2 mmol) and stirredunder nitrogen to give a deep yellow solution. To this was addedO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate(3.5 g, 9 mmol) and stirring continued for 2 hrs. During this time athick yellow precipitate formed. 6-Aminohexanoic acid (1.45 g, 11 mmol)was then added and stirring continued overnight. Reverse phasechromatographic analysis (C₁₈) (methanol:water, 80:20) indicated twospots at R_(f)=0.75 and R_(f)=0.55. The reaction mixture was then pouredinto 0.25M aqueous HCl (200 ml) and the precipitated product collectedby vacuum filtration, washing with more dilute HCl and water. Thestill-damp solid was recrystallized from ethanol/water and dried undervacuum over phosphorus pentoxide to give6-(9-oxo-9H-acridin-4-carboxamido)hexanoic acid (1.97 g, 62%). λ_(max)(EtOH)=408, 390, 256 nm. δ_(H) (300 MHz, DMSO-d₆) 1.35-1.65 (6H, m),2.24 (2H, t), 3.40 (2H, t), 7.26-7.39 (2H, m), 7.74-7.77 (2H, m),8.21-8.28 (2H, m), 8.44 (2H, app.d), 9.00 (1H, broad t, amide), 12.02(broad s, D₂O exch.) and 12.49 (broad s, D₂O exch.). Mass spectrum:MALDI-TOF, m/z=353.15, M=353.15 for O₂₀H₂₁N₂O₄. Melting Point=218° C.

4.3 O—(N-Succinimidyl)-6-(9-oxo-9H-acridin-4-carboxamido)hexanoate

6-(9-Oxo-9H-acridin-4-carbaxamido)hexanoic acid (352 mg, 1.0 mmol) andO—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TSTU;301 mg, 1.0 mmol) were dissolved in N,N-dimethylformamide (5 ml). To theresulting solution was added N,N-diisopropylethylamine (183 μl, 1.05mmol). After leaving to stand overnight the solvent was evaporated undervacuum. The residue was purified by flash chromatography (silica.10-100% ethyl acetate/dichloromethane) to giveO—(N-succinimidyl)-6-(9-oxo-9H-acridin-4-carbaxamido)hexanoate (410 mg,91%). δ_(H) (200 MHz, DMSO-d₆) 1.44-1.75 (6H, m), 2.71 (2H, t), 2.79(4H, s), 3.38 (2H, t), 7.27-7.39 (2H, m), 7.75 (2H, app.d), 8.21-8.28(2H, m), 8.44 (1H, app.d), 9.00 (1H, broad t, amide) and 12.47 (broad s,D₂O exch.). Mass spectrum: (ES+) 450 (M+H), 472 (M+Na). Accurate mass:(M+H)=C₂₄H₂₄N₃O₆, requires 450.1665. Found 450.1671 (+1.3 ppm).

5. 2-Carboxymethyl-7-chloro-9-oxo-9,10-acridine

5.1 N-(4-Carboxymethylphenyl)-4-chloro-2-carboxyaniline

To a 100 ml round bottomed flask was added 2,5-dichlorobenzoic acid (1.9g, 10 mmol), 4-aminophenylacetic acid (1.5 g, 10 mmol), anhydrous sodiumcarbonate (3.2 g, 26 mmol), activated copper metal powder (0.25 g, 4mmol) and 1-butanol (50 ml). The flask was fitted with a magneticstirrer bar, water condenser, silica gel guard tube and heated underreflux for 48 hours. TLC (RPC₁₈, Water, 20: methanol, 80) showed theformation of a slower moving component at R_(f) 0.55. The solvent wasremoved under reduced pressure with final drying under high vacuum. Theresidue was dissolved in 50 ml water and heated to boiling, thencharcoal was added and the mixture filtered through celite, washingthrough with a further 25 ml of hot water. This solution was cooled to10° C. in an ice bath and then acidified to pH≈2 with to concentratedaqueous HCl. The oil that separated was extracted into chloroform, thesolution dried with anhydrous magnesium sulphate, filtered and thesolvent removed by rotary evaporation to leave a sticky solid.Recrystallization from water/acetic acid gave the title compound (1.07g, 35%). δ_(H) (300 MHz, DMSO-d₆) 3.54 (2H, s), 7.15-7.27 (5H, m), 7.39(1H, dd) 7.81 (1H, d), 9.54 (1H, broad s) and 12.0-13.5 (2H, broad).Mass spectrum: (ES−) 304 (M−H). λ_(max) (EtOH)=292, 364 nm.

5.2 2-Carboxymethyl-7-chloro-9-oxo-9,10-acridine

To a 25 ml round bottomed flask was added the diphenylamine (500 mg,1.64 mmol) and phosphorus oxychloride (5 ml). The flask was fitted witha magnetic stirrer bar, water condenser, silica gel guard tube andheated under reflux for 1 hour. The excess phosphorus oxychloride wasremoved from the dark brown mixture under vacuum, then a small amount ofice was added followed by 2.0M aqueous HCl (10 ml). The mixture washeated to 100° C. for 1 hour and allowed to cool, before beingevaporated to dryness and dried under vacuum over phosphorus pentoxide.The residue was dissolved in 10% v/v water/methanol and eluted through aSepPak RPC₁₈ (10 g) column with monitoring by TLC. Thefluorescence-containing fractions were combined, evaporated to drynessand dried under vacuum over phosphorus pentoxide.

This semi-purified material was then further purified by preparativeHPLC(RPC₁₈. Water→methanol gradient). Pure fractions were combined andevaporated to give 2-carboxymethyl-7-chloro-9-oxo-9,10-acridine as apale yellow solid (19 mg, 4%). δ_(H) (300 MHz, DMSO-d₆) 3.72 (2H, s),7.50 (1H, m), 7.58 (1H, m), 7.65 (1H, m), 7.75 (1H, m), 8.10 (1H, m),8.14 (1H, m) and 11.9+12.4 (2× broad s). Mass spectrum: (ES−) 286+288(M−H), 242+244 (M−H—CO₂). λ_(max) (EtOH)=260, 390, 408 nm.

6. N-(Succinyl)-2-amino-10H-acridine-9-one

2-Amino-10H-acridine-9-one (100 mg, 0.48 mM), succinic anhydride (50 mg,0.5 Mm), diisopropylethylamine (90 μl) and dry DMF (1 ml) were stirredtogether overnight. TLC (C-18 reverse phase plates, water:methanol 1:1)indicated that all the starting material (green fluorescence under longwavelength UV light) had been converted to a faster running spot, whichshowed blue fluorescence under long wavelength UV light. The solvent wasremoved by rotary evaporation, the residue dissolved in dichloromethaneand purified by flash column chromatography (silica, 0-10%methanol/dichloromethane). Pure fractions of each were pooled andevaporated to dryness to give N-(succinyl)-2-amino-10H-acridine-9-one,147 mg (98%). λ_(max) (H₂O)=399 nm. δ_(H) (300 MHz, d₆-DMSO) 2.05 (4H,m), 6.82 (1H, t), 7.15 (2H, m), 7.28 (1H, t), 7.53 (1H, d), 7.81 (1H,d), 8.09 (1H, s). Mass spectrum: (ES+) 311. M.Pt>300° C.

7. 2-Nitroacridone-7-sulphonic acid

7.1 2-Carboxy-4-nitrodiphenylamine

2-Chloro-5-nitrobenzoic acid (15 g, 75 mmol) was mixed with 1-butanol(100 ml) and stirred. To the resulting mixture was added aniline (15 ml,15.3 g, 165 mmol) and N,N-diisopropylethylamine (28.8 ml, 21.3 g, 165mmol). The resulting light yellow solution was heated under reflux for 4days, during which time the colour changed to deep yellow. TLC (RPC₁₈,Methanol, 80:water, 20. R_(f)=0.75, yellow: R_(f)2-chloro-5-nitrobenzoic acid=0.85).

The solvent was evaporated under vacuum as much as possible. Theresidual oil was triturated with water acidified with aqueous HCl tomaintain a pH of 2-4. A yellow solid eventually separated. This wascollected by vacuum filtration, washed well with excess water andallowed to suck dry over 15 minutes. The crude product was purified bymixing with acetic acid (150 ml), then heating to boiling and allowingthe resulting mixture to cool slowly to ambient temperature withstirring. The bright yellow solid so obtained was collected by vacuumfiltration, washed with acetic acid followed by excess diethyl ether anddried under vacuum over phosphorus pentoxide to give2-carboxy-4-nitro-diphenylamine (12.02 g, 62%). δ_(H) (200 MHz, DMSO-d₆)7.14 (1H, d), 7.24-7.52 (5H, m), 8.18 (1H, dd), 8.72 (1H, d) and 10.38(1H, broad s).

7.2 2-Nitroacridone

2-Carboxy-4-nitrodiphenylamine (5.16 g, 20 mmol) was mixed withphosphorus oxychloride (20 ml). The resulting yellow slurry was heatedunder reflux for 3 hrs, during which time the solids dissolved to give adark solution (intensely yellow at the meniscus). The excess phosphorusoxychloride was then evaporated under vacuum; the resulting oil wascarefully quenched with ice before addition of 1.0M aqueous HCl (100ml). The mixture was then heated to boiling, whereupon acetic acid (upto 20 ml) was slowly added down the condenser to aid dispersion of theimmiscible oil. On continued boiling for 1 hour, a yellow slurryresulted. After subsequent cooling, this solid was collected by vacuumfiltration, washed with excess water, then acetone and finally diethylether, before drying under vacuum over phosphorus pentoxide to give2-nitroacridone (4.66 g, 97%). δ_(H) (200 MHz, DMSO-d₆) 7.39 (1H,app.t), 7.60 (1H, d), 7.68 (1H, d), 7.84 (1H, app.t), 8.25 (1H, d), 8.48(1H, dd), 8.98 (1H, d) and 12 38 (1H, broad s).

7.3 2-Nitroacridone-7-sulphonic acid

2-Nitroacridone (25 mg) was mixed with fuming sulphuric acid (˜20% freeSO₃, 2.5 ml) to give a reddish solution. This was heated at 100° C. for90 mins, to give a yellowish solution. After cooling, the mixture wasquenched dropwise into ice (˜15 g); 6 ml of concentrated HCl were addedand the mixture left to stand to precipitate out the product. Theresulting pale yellow solid was collected by vacuum filtration, washedwith a little 4.0M aqueous HCl, then redissolved in water and filteredinto a clean flask. The water was evaporated under vacuum to leave2-nitroacridone-7-sulphonic acid as a yellow-brown solid. TLC acid(RPC₁₈, Methanol, 80: water, 20. R_(f)=0.8, yellow. Mass spectrum: (ES+)321 (M+H); accurate mass: (M+H)=C₁₃H₃N₂O₆S requires 321.0181. Found321.0187 (1.8 ppm).

8. O—(N-Succinimidyl)-6-(2-acetamido)-9-oxo-9H-acridin-10-yl)hexanoate

8.1 O-Ethyl-6-(2-nitro-9-oxo-9H-acridin-10-yl)hexanoate

2-Nitroacridone (2.4 g; 10 mmol) was stirred with anhydrous methylsulphoxide (25 ml) under a nitrogen atmosphere. After 5 minutes, sodiumhydride (60% dispersed in oil, 480 mg; 12 mmol) was added to the yellowsolution. Stirring was continued for 90 mins. during which time thesolution turned magenta. Ethyl 6-bromohexanoate (2.67 ml; 12 mmol) wasadded and stirring continued overnight. The reaction mixture was pouredinto water (300 ml) and the yellow precipitate was collected byfiltration, washed with water and dried under vacuum. The solid wasdissolved in dichloromethane and anhydrous magnesium sulphate added tothe solution. After filtration, the solution was evaporated to drynessto leave a yellow-brown solid. The crude product was purified by flashchromatography (silica. 0-5% ethyl acetate/dichloromethane) to give 1.19g (50%) of O-ethyl-6-(2-nitro-9-oxo-9H-acridin-10-yl)hexanoate.

δ_(H) (200 MHz, DMSO-d₆) 1.2 (3H, t), 1.7 (6H, m), 2.3 (2H, t), 4.05(2H, q), 4.55 (2H, m), 7.45 (1H, m), 7.92 (2H, d), 8.03 (1H, d), 8.35(1H, d), 8.50 (1H, dd), 9.03 (1H, d).

8.2 O-Ethyl-6-(2-amino-9-oxo-9H-acridin-10-yl)hexanoate

O-Ethyl-6-(2-nitro-9-oxo-9H-acridin-10-yl)hexanoate (1.91 g; 5.0 mmol)and ammonium formate (1.58 g; 25 mmol) were dissolved in ethanol (100ml) to give a yellow solution. The solution was stirred under nitrogenand a catalytic amount of 5% palladium on carbon was added. Stirring wascontinued for 5 hrs. The solution was then filtered through celite andthe solvent removed by rotary evaporation. The residue was dissolved indichloromethane and extracted with water. The organic layer was driedwith anhydrous magnesium sulphate, filtered and the solvent removed byrotary evaporation. The crude product was purified by flashchromatography (silica. 4-6% methanol/dichloromethane) to give 1.66 g

(94%) of O-ethyl-6-(2-amino-9-oxo-9H-acridin-10-yl)hexanoate. δ_(H) (200MHz, DMSO-d₆) 1.15 (3H, t), 1.6 (6H, m), 2.35 (2H, t), 4.05 (2H, dd),4.4 (2H, t), 5.3 (2H, s), 7.2 (2H, m), 7.6 (4H, m), 8.3 (1H, d).

8.3 6-(2-Amino-9-oxo-9H-acridin-10-yl)hexanoic acid

O-Ethyl-6-(2-amino-9-oxo-9H-acridin-10-yl)hexanoate (350 mg; 1.0 mmol)was dissolved in acetic acid (5 ml) and 1.0M hydrochloric acid (2 ml)and refluxed for 4 hrs. The solvent was removed by rotary evaporation,the residue dissolved in acetic acid and evaporated to dryness and theprocess repeated twice using acetonitrile as solvent. The residue wasdried under vacuum to give 370 mg of6-(2-amino-9-oxo-9H-acridin-10-yl)hexanoic acid.

8.4 6-(2-Acetamido-9-oxo-9H-acridin-10-yl)hexanoic acid

6-(2-Amino-9-oxo-9H-acridin-10-yl)hexanoic acid (370 mg: 1.14 mmol) wasdissolved in anhydrous pyridine (10 ml) and acetic anhydride (100 μl)followed by diisopropylethylamine (350 μl). The mixture was stirred for3 hours. The solution was evaporated to dryness under vacuum and thegummy residue dissolved in dichloromethane. The solution was washed with1.0M hydrochloric acid and then brine. The organic phase was dried withanhydrous magnesium sulphate, filtered and the solvent removed by rotaryevaporation to leave a sticky solid. Trituration with ether gave a solidwhich was dried under vacuum to give 360 mg (86%) of6-(2-acetamido-9-oxo-9H-acridin-10-yl)hexanoic acid.

8.5 O—(N-Succinimidyl)-6-(2-acetamido-9-oxo-9H-acridin-10-yl)hexanoate

6-(2-Acetamido-9-oxo-9H-acridin-10-yl)hexanoic acid (360 mg; 1.0 mmol)and O—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(350 mg; 1.0 mmol) were dissolved in anhydrous dimethylformamide (5 ml)and diisopropylethylamine (183 μl) added. The yellow solution wasstirred under nitrogen for 1 hour. The solvent was removed by rotaryevaporation to leave a brown gum. This was purified by flashchromatography (silica. 10% methanol/ethyl acetate) to give 330 mg (50%)of O—(N-succinimidyl)-6-(2-acetamido-9-oxo-9H-acridin-10-yl)hexanoate.δ_(H) (200 MHz, DMSO-d₆) 1.8 (6H, m), 2.1 (3H, s), 2.9 (6H, m), 4.5 (2H,m), 7.3 (1H, m), 7.8 (3H, m), 8.05 (1H, d), 8.35 (1H, d), 8.6 (1H, s),10.2 (1H, s). Accurate mass. (M+H)=C₂₅H₂₆N₃O₆, requires 464.1822. Found464.1798 (5.1 ppm).

9. O—(N-Succinimidyl)-6-(2-sulpho-9-oxo-9H-acridin-10-yl)hexanoate

9.1 6-(2-Sulpho-9-oxo-9H-acridin-10-yl)hexanoic acid

O-Ethyl-6-(9-oxo-9H-acridin-10-yl)hexanoate (2.0 g; 6.0 mmol) wasdissolved in conc. sulphuric acid (10 ml) and the solution heated to120° C. for 20 hrs. The mixture was allowed to cool and added to ˜50 gmof crushed ice. The precipitate was collected by centrifugation, washedwith 3.0M hydrochloric acid and dried under vacuum and over phosphorouspentoxide to give 2.1 g (90%) of6-(2-sulpho-9-oxo-9H-acridin-10-yl)hexanoic acid. δ_(H) (200 MHz,DMSO-d₆) 1.7 (6H, m), 2.26 (2H, t), 4.5 (2H, t), 7.37 (1H, m), 7.9 (5H,m), 8.37 (1H, d), 8.58 (1H, d). Mass spectrum: (ES+) (M+H) 390.

9.2 O—(N-Succinimidyl)-6-(2-sulpho-9-oxo-9H-acridin-10-yl)hexanoate

6-(2-Sulpho-9-oxo-9H-acridin-10-yl)hexanoic acid (100 mg; 0.25 mmol) wasdissolved in anhydrous dimethylformamide (3 ml) and evaporated todryness on a rotary evaporator. The process was repeated to removetraces of water. O—(N-Succinimidyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (90 mg; 0.3 mmol) was added and the mixture dissolvedin anhydrous dimethylformamide (2 ml) and diisopropylethylamine (90 μl).The yellow solution was stirred under nitrogen for 60 mins when TLC(RP₁₈ 50:50 water:methanol) showed all the starting material had beenconverted to a slower moving component. The solvent was removed byrotary evaporation with final drying under high vacuum. No furtherattempts were made to purify this material.

10. 6-(2-Bromo-7-sulpho-9-oxo-9H-acridin-10-yl)hexanoic acid

O-ethyl-6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoate (2.08 gm; 5.0 mmol)was dissolved in conc. sulphuric acid (10 ml) and heated to 120° C. for20 hrs. The mixture was allowed to cool and added to ˜50 gm of crushedice. The precipitate was collected by centrifugation, washed with 3.0Mhydrochloric acid and dried under vacuum in the presence of phosphorouspentoxide to give 2.2 g (94%) of6-(2-bromo-7-sulpho-9-oxo-9H-acridin-10-yl)hexanoic acid. δ_(H) (200MHz, DMSO-d₆) 1.7 (6H, m), 2.25 (2H, t), 4.5 (2H, m), 7.9 (4H, m), 8.42(1H, d), 8.57 (1H, d). Mass spectrum (ES+) (M+H) 468, 470.

11. N-(Maleimido)ethyl-6-(9-oxo-9H-acridin-10-yl)hexanamide

11.1 N-(Aminoethyl)maleimide hydrochloride

N-Butoxycarbonyl-2-(aminoethyl)maleimide (200 mg; 0.83 mmol) was stirredunder nitrogen with 4M hydrochloric acid in dioxan (4 ml). A whiteprecipitate started to form after a few minutes. Stirring was continuedfor 40 minutes and then the solvent was removed by rotary evaporation.The resultant white solid was dried under vacuum to give 180 mg (100%)of N-(aminoethyl)maleimide hydrochloride. δ_(H) (200 MHz, CD₃OD) 1.38(2H, s), to 3.14 (2H, t), 3.81 (2H, t), 6.90 (2H, s).

11.2 N-(Maleimido)ethyl-6-(9-oxo-9H-acridin-10-yl)hexanamide

O—(N-Succinimidyl)-6-(9-oxo-9H-acridin-10-yl)hexanoate (102 mg; 0.25mmol) was dissolved in anhydrous dimethyl formamide (800 μl) anddiisopropylethylamine (53 μl) added. N-(Aminoethyl)maleimidehydrochloride (53 mg; 0.30 mmol) was added to the yellow solution whichwas left to stand overnight. The solvent was removed by rotaryevaporation and the residue purified by flash chromatography (silica. 2%methanol/dichloromethane). After removal of the solvent by rotaryevaporation a yellow oil remained which slowly crystallised. Triturationwith diethyl ether completed the crystallisation. This material wasfurther purified by preparative TLC (silica. 5%methanol/dichloromethane) extracting the required material with 10%methanol/dichloromethane.

Solvent was removed under vacuum, the residue triturated with ether togive 65 mg (60%) ofN-(maleimido)ethyl-6-(9-oxo-9H-acridin-10-yl)hexanamide. δ_(H) (200 MHz,DMSO-d₆) 1.7 (6H, m), 2.02 (2H, t), 3.2 (2H, dd), 3.45 (2H, t), 4.45(2H, t), 7.01 (2H, s), 7.35 (2H, m), 7.85 (5H, m), 8.36 (2H, d). Massspectrum (ES+) (M+H) 432.

12. N-(Maleimido)ethyl-6-(2-sulpho-9-oxo-9H-acridin-10-yl)hexanamide

O—(N-Succinimidyl)-6-(2-sulpho-9-oxo-9H-acridin-10-yl)hexanoate (90 mg;0.15 mmol) was dissolved in anhydrous dimethyl formamide (1.0 ml) anddiisopropylethylamine (52 μl) added. N-(aminoethyl)maleimidehydrochloride (52 mg; 0.30 mmol) was added to the yellow solution whichwas is left to stand overnight. The solvent was removed by rotaryevaporation and the residue purified by HPLC (Vydac RP₁₈semi-preparative column, gradient of water to 25% acetonitrile (bothcontaining 0.1% trifluoroacetic acid) over 30 minutes, detection at 400nm) to giveN-(maleimido)ethyl-6-(2-sulpho-9-oxo-9H-acridin-10-yl)hexanamide.

13. 6-(2-Fluoro-9-oxo-9H-acridin-10-yl)hexanoic acid

13.1 N-(4-Fluorophenyl)anthranilic acid

4-Fluoroaniline (1.86 gm; 20 mmol), 2-chlorobenzoic acid (1.56 gm; 10mmol), ethylene glycol (5 ml) and anhydrous sodium carbonate (1.1 gm; 10mmol) were placed in a reaction vessel and stirred until effervescenceceased. Cupric chloride (100 mg; 0.75 mmol) dissolved in 2 ml of waterwas added to the reaction mixture which was then heated to 125° C. for 6hrs. The reaction was allowed to cool and water (30 ml) and charcoalwere added. The mixture was filtered and then acidified to pH 2 withconc. hydrochloric acid. The precipitate was collected by filtration,washed with water and then re-dissolved in 1M sodium hydroxide solution.Material was re-precipitated by the addition of acetic acid, filteredoff, washed with aqueous acetic acid, then water and finally dried undervacuum over phosphorous pentoxide to give 862 mg (37%) ofN-(4-fluorophenyl)anthranilic acid.

13.2 2-Fluoroacridone

N-(4-Fluorophenyl)anthranilic acid (0.70 gm; 3 mmol) and phosphorousoxychloride (3 ml) were stirred together and heated to 115° C. for 3.5hours, is then allowed to cool. The reaction mixture was placed on iceand small pieces of ice added, a vigorous reaction occurred with theevolution of hydrogen chloride. When the reaction had subsided, water(15 ml) was added and the mixture was boiled for 2 hours. On cooling, asolid precipitated out. This was filtered off and washed with wateruntil the filtrate was colourless. The precipitate was further washedwith cold methanol then diethyl ether and dried under vacuum to give 383mg (59%) of 2-fluoroacridone.

13.3 O-Ethyl-6-(2-fluoro-9-oxo-9H-acridin-10-yl)hexanoate

2-Fluoroacridone (213 mg; 1.0 mmol) was dissolved in anhydrous DMF (3ml) under a nitrogen atmosphere. Sodium hydride dispersed in oil (45 mg;1.1 mmol) was added and the mixture stirred until effervescence ceased.Ethyl 6-bromoacetate (250 μl) was added and the mixture stirred at 70°C. overnight. The solvent was removed by rotary evaporation and theyellow residue purified by flash chromatography (silica. 4% ethylacetate/dichloromethane) to give 230 mg (65%) ofO-ethyl-6-(2-fluoro-9-oxo-9H-acridin-10-yl)hexanoate. δ_(H) (200 MHz,DMSO-d₆) 1.20 (3H, t), 1.65 (6H, m), 2.35 (2H, t), 4.05 (2H, dd), 4.45(2H, t), 7.35 (1H, m), 7.9 (5H, m), 8.35 (1H, d). Mass spectrum (ES+)(M+H) 356.1

13.4 6-(2-Fluoro-9-oxo-9H-acridin-10-yl)hexanoic acid

O-Ethyl-6-(2-fluoro-9-oxo-9H-acridin-10-yl)hexanoate (71 mg; 0.2 mmol)was dissolved in ethanol (2 ml) and 1.0M sodium hydroxide solution (0.4ml) added and the mixture heated to 90° C. for 90 minutes. The mixturewas cooled and water (6 ml) added to give a yellow precipitate. Themixture was to cooled on ice and acidified with conc. hydrochloric acidwhen more material precipitated out. The precipitate was filtered off,washed with water then ethanol and dried under vacuum over phosphorouspentoxide to give 47 mg (72%) of6-(2-fluoro-9-oxo-9H-acridin-10-yl)hexanoic acid. δ_(H) (200 MHz,DMSO-d₆) 1.68 (6H, m), 2.25 (2H, t), 4.48 (2H, t), 7.36 (1H, m), 7.85(5H, m), 8.34 (1H, d).

λ_(max)(ab) 251 nm (ε=45,500/M⁻¹ cm⁻¹); 401 nm (ε=7980/M⁻¹ cm⁻¹); 421 nm(ε=7980/M⁻¹ cm⁻¹). (PBS buffer). λ_(max)(em) 434 nm (PBS buffer)

14. 6-(2-Methoxy-9-oxo-9H-acridin-10-yl)hexanoic acid

14.1 N-(4-Methoxyphenyl)anthranilic acid

4-Methoxyaniline (1.86 gm; 20 mmol), 2-chlorobenzoic acid (1.56 gm; 10mmol), ethylene glycol (5 ml) and anhydrous sodium carbonate (1.1 gm; 10mmol) were placed in a reaction vessel and stirred until effervescenceceased. Cupric chloride (100 mg; 0.75 mmol) dissolved in 2 ml of waterwas added to the reaction mixture which was then heated to 125° C. for 6hours. The reaction was allowed to cool then water (30 ml) and charcoalwere added. The mixture was filtered and acidified to pH 2 with conc.hydrochloric acid. The precipitate was collected by filtration, washedwith water and then re-dissolved in 1M sodium hydroxide solution.Material was re-precipitated by the addition of acetic acid, filteredoff, washed with aqueous acetic acid, then water and finally dried undervacuum over phosphorous pentoxide to give 1200 mg (49%) ofN-(4-methoxyphenyl)anthranilic acid. Mass spectrum (ES+) (M+H) 242

14.2 2-Methoxyacridone

Polyphosphoric acid (50 gm) was heated to 160° C. under a nitrogenatmosphere. N-(4-methoxyphenyl)anthranilic acid (4.89 gm; 20 mmol) wasadded and the mixture stirred at 160° C. for 15 minutes. The reactionwas is cooled rapidly in an ice bath and water added to give a greenishyellow precipitate. This was filtered off, washed with water, thendilute sodium bicarbonate solution and again with water. The solid wasfinally dried at 50° C. under vacuum to give 3.67 gm (81%) of2-methoxyacridone. δ_(H) (200 MHz, DMSO-d₆) 3.86 (3H, s), 7.23 (1H, t),7.55 (5H, m), 8.22 (1H, d), 11.7 (1H, s).

14.3 O-Ethyl-6-(2-methoxy-9-oxo-9H-acridin-10-yl)hexanoate

2-Methoxyacridone (2.25 g; 10 mmol) was stirred with anhydrous dimethylformamide (15 ml) under a nitrogen atmosphere. After 5 minutes, sodiumhydride (60% dispersed in oil, 250 mg; 12 mmol) was added and themixture stirred until effervescence ceased. A second lot of sodiumhydride (230 mg) was added and stirring continued until effervescenceceased. Ethyl 6-bromohexanoate (2.67 ml; 15 mmol) was added to theyellow solution and stirring was continued overnight. The reactionmixture was poured into water (300 ml) and the mixture extracted withdichloromethane. The organic phase was washed with 1.0M hydrochloricacid (2×150 ml) then dried over anhydrous magnesium sulphate. Afterfiltration, the solvent was removed by rotary evaporation to give a darkcoloured oil. This was purified by flash chromatography (silica. 5%ethanol/dichloromethane) to give a yellow oil which crystallised ontrituration with diethyl ether/hexane to give 0.83 g (23%) ofO-ethyl-6-(2-methoxy-9-oxo-9H-acridin-10-yl)hexanoate. Mass spectrum(ES+) (M+H) 367 (M+Na) 389.

14.4 6-(2-Methoxy-9-oxo-9H-acridin-10-yl)hexanoic acid

O-Ethyl-6-(2-methoxy-9-oxo-9H-acridin-10-yl)hexanoate (367 mg; 1.0 mmol)was dissolved in ethanol (10 ml) and 1.0M sodium hydroxide solution (2.0ml) added and the mixture heated to 90° C. for 1 hour. The mixture wascooled and water (20 ml) added. The mixture was cooled on ice andacidified with conc. hydrochloric acid when a yellow oil separated. Theoil slowly crystallised to a bright yellow solid. This was filtered off,washed with water and dried under vacuum to give 327 mg (96%) of6-(2-methoxy-9-oxo-9H-acridin-10-yl)hexanoic acid. δ_(H) (200 MHz,DMSO-d₆) 1.7 (6H, m), 2.25 (2H, t), 3.9 (3H, s), 4.5 (2H, t), 7.31 (1H,m), 7.48 (1H, dd), 7.82 (4H, m), 8.35 (1H, d). λ_(max)(ab) 255 nm(ε=38,100/M⁻¹ cm⁻¹); 408 nm (ε=7150/M⁻¹ cm⁻¹); 428 nm (ε=7150/M⁻¹ cm⁻¹).(PBS buffer). λ_(max)(em) 467 nm. (PBS buffer).

15. Fluorescence Lifetime Studies

Fluorescence lifetimes were determined by time-correlated single photoncounting using an Edinburgh Instruments FL900CDT Time ResolvedT-Geometry Fluorimeter. Samples were excited at 400 nm using a hydrogenarc lamp. Detection was at 450 nm. Deconvolution using a non-linearleast squares algorithm gave the results shown in Table 2. FIG. 2 is aplot showing the fluorescence lifetimes of certain acridone dyesaccording to the invention.

TABLE 2 Fluorescence Lifetimes Lifetime Compound Solvent (nsecs)N-(Succinyl)-2-amino-10H-acridine-9-one water 17.22-Carboxymethyl-7-chloro-9-oxo-9,10-acridine MeOH 16.86-(2,7-Disulphonato-9-oxo-9H-acridin-10-yl)hexanoic water 14.6 acid6-(9-Oxo-9H-acridin-10-yl)hexanoic acid water/ 14.2 MeOH 50/50)6-(2-Bromo-9-oxo-9H-acridin-10-yl)hexanoic acid MeOH 8.36-(2,7-Dibromo-9-oxo-9H-acridin-10-yl)hexanoic acid MeOH 4.56-(9-Oxo-9H-acridin-4-carboxamido)hexanoic acid water/ 4.2 MeOH (50/50)2-Nitroacridone-7-sulphonic acid water non- fluorescent6-(2-Acetamido-9-oxo-9H-acridin-10-yl)hexanoic acid water 176-(2-Sulpho-9-oxo-9H-acridin-10-yl)hexanoic acid water 13.36-(2-Bromo-7-sulpho-9-oxo-9H-acridin-10-yl)hexanoic water 5.6 acid6-(2-Fluoro-9-oxo-9H-acridin-10-yl)hexanoic acid water 146-(2-Methoxy-9-oxo-9H-acridin-10-yl)hexanoic acid water 17

16. Protein Labelling 16.1 Preparation of6-(9-oxo-9H-acridin-10-yl)hexanoic acid—bovine serum albumin (BSA)conjugate (Conjugate 1)

To 10 ml of bovine serum albumin (1 mg/ml in 0.1 M carbonate buffer,pH9.3), 100 μl O—(N-succinimidyl)-6-(9-oxo-9H-acridin-10-yl)hexanoate (1mg/100 μl in DMSO) was added dropwise whilst stirring. Gentle stirringcontinued for 1 hr at ambient temperature in a foil wrapped vial.Unconjugated dye was removed by overnight dialysis (12-14K MWCO) at 4°C. with at least 2 is changes of PBS. Conjugate 1 was recovered andstored at 4° C.

16.2 Preparation of 6-(9-oxo-9H-acridin-4-carboxamido)hexanoicacid—rabbit serum albumin conjugate (Conjugate 2)

To 10 ml of rabbit serum albumin (1 mg/ml in 0.1 M carbonate buffer,pH9.3), 100 μlO—(N-succinimidyl)-6-(9-oxo-9H-acridin-4-carboxamido)hexanoate (1 mg/100μl in DMSO) was added dropwise whilst stirring. Gentle stirringcontinued for 1 hr at ambient temperature in a foil wrapped vial.Unconjugated dye was removed by overnight dialysis (12-14K MWCO) at 4°C. with at least 2 changes of PBS. Conjugate 2 was recovered and storedat 4° C.

16.3 Determination of the Fluorescence Lifetimes of Conjugates 1 and 2

The fluorescence lifetimes of a mixture of conjugates 1 and 2 weredetermined in PBS. The results are shown in FIG. 3. Deconvolution andcurve fitting using a non-linear least-squares algorithm gave theresults shown in Table 3.

TABLE 3 Lifetime Relative Sample (ns) % Conjugate 1 14.0 40.2 5.5 45.61.2 14.3 Conjugate 2 4.6 49.4 1.2 35.0 19.3 15.6

16.4 Immunoprecipitation Assay

To 500 μl of PBS in a 1.5 ml centrifuge tube was added 200 μl conjugate1 and 200 μl conjugate 2. After mixing, 450 μl was removed into a silicacuvette for determination of the fluorescence lifetime using excitationat 405 nm, emission at 450 nm by a time-correlated single photoncounting technique (Edinburgh Analytical Instruments FL900CDTspectrometer). To the 450 μl in the centrifuge tube, 100 μl of anti-BSAantibody was added. After incubation for 30 min at 37° C., then 1 hrincubation at 4° C., the tube was centrifuged for 5 min in a bench-topcentrifuge. The pellet was washed twice with ice-cold PBS, thenre-suspended in 0.1 M NaOH. The fluorescence lifetime of this solutionwas determined as above. The results are shown in FIG. 4. Deconvolutionand curve fitting using a non-linear least-squares analysis algorithmgave the results shown in Table 4.

TABLE 4 Lifetime Fits for Immunoprecipitation Assay Lifetime RelativeSample (ns) % Initial mixture 14.7 33.3 5.0 47.5 0.9 19.2 Re-suspendedpellet 13.1 70.3 4.7 22.8 0.7 6.9

The results show that the relative percentage of conjugate 1 (lifetimerange 13-15 ns) has increased significantly in the pellet, as a resultof the immunoprecipitation by the anti-BSA antibody. The proportion ofconjugate 2 (lifetime range 4.5-5 ns) in the re-suspended pellet iscorrespondingly decreased, relative to the proportion in the initialmixture. Although the immunoprecipitation process was not completelyefficient, analysis using fluorescence lifetime has enabled theresolution of two species (conjugate 1 and conjugate 2) in mixtureswhich are indistinguishable by their emission wavelength.

17. Fluorescence Lifetime Detection in Capillary Electrophoresis ofAcridone Dye-Labelled DNA Fragments

M13 DNA primers were labelled using standard techniques with each offour acridone dyes according to the present invention, i.e:

-   i) 6-(2-(acetylamino)-9-oxo-9H-acridin-10-yl)hexanoic acid,-   ii) 6-(9-oxo-9H-acridin-10-yl)hexanoic acid,-   iii) 6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoic acid, and-   iv) 6-(9-oxo-9H-acridin-4-carboxamido)hexanoic acid.    The succinimidyl ester of each dye was conjugated to an    amine-modified M13 forward sequencing primer in 0.1 M carbonate pH    9.3/DMF (final composition 2:1). Purification by HPLC on a C18    column used a triethylammonium bicarbonate/MeCN solvent system.

Real-time fluorescence lifetime detection was achieved by interfacing acommercial multiharmonic Fourier transform (MHF) fluorescence lifetimeinstrument (Model 4850 MHF, Spectronics Instruments, Rochester, N.Y.) toa Beckman P/ACE 5000 CE system (Li, L. et al, J. Chromatogr. B, (1997),695, 85-92). The excitation source was a continuous wave violet diodelaser that supplied 25-30 mW at 405 nm. The laser beam was focused ontothe detection window of the capillary using either a 45 mm focusing lensor a 6.3× microscope objective with a focal length of 22 mm. Theemission signal was collected by a 40× microscope objective. Emissionwas selected through a 435 nm long pass filter. A cross-correlationfrequency of 9.4 Hz was used in the lifetime measurements, resulting in9.4 phase and modulation measurements per second. Ten successivemeasurements were then averaged prior to data analysis to yieldapproximately one lifetime measurement per second. Scattered light fromthe capillary provided the lifetime reference.

Solutions of 0.5 mM dye-labelled primer in 100 mM Tris buffer, pH 8.6,were injected into the bare 75 μM internal diameter capillary for 10 seach at 10 kV injection voltage. The separation voltage was 18 kV (250V/cm). The replaceable gel matrix contained 2% POP-6 gel in 3.5×POP-6buffer. The lifetime electropherogram as shown in FIG. 5 was obtainedfor successive injections of M13 DNA primers labelled with each of thefour dyes. The solid line is the intensity and the dots correspond tolifetimes recovered from a 1-component fit using non-linear leastsquares analysis software (Globals, Inc). The results show that thefluorescence lifetime (dots) coincides with the fluorescence intensitypeaks (line) as the dye labelled M13 primers migrate past the detector.

18. Multiplexing Fluorescence Lifetime Determination

The following fluorescent acridone dye derivatives were prepared as 1mg/ml stock solutions in methanol:

-   a) 6-(9-oxo-9H-acridin-10-yl)hexanoic acid;-   b) 6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoic acid; and-   c) 6-(9-oxo-9H-acridin-4-carboxamido)hexanoic acid.

The methanolic stock solutions were diluted (1/100) into 12%polyacrylamide mix which was allowed to polymerize directly indisposable cuvettes. Mixtures of the dyes were similarly prepared. Thefluorescence lifetimes of samples containing single, or mixtures of dyesin polyacrylamide gel, were recorded by a time-correlated single photoncounting technique (Edinburgh Analytical Instruments FL900CDTspectrometer). Samples were excited at 400 nm using a hydrogen arc lamp,detection was at 450 nm. Deconvolution using a non-linear least squaresalgorithm gave the results shown in Table 5.

TABLE 5 Principal Lifetime Relative Sample (ns) % a)6-(9-oxo-9H-acridin-10-yl)hexanoic acid 14.0 96.8 b)6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoic acid 9.0 77.1 c)6-(9-oxo-9H-acridin-4-carboxamido)hexanoic 3.6 74.4 acid a) + c) 14.074.5 3.9 25.5 b) + c) 9.5 49.7 4.0 50.3 a) + b) + c) 14.3 55.5 7.9 26.73.5 17.9

The results show that multiple lifetime components are reported when thedyes are analysed in PAGE. However, the principal lifetime component ofeach of the single dyes can still be distinguished in mixtures of thedyes in PAGE. Thus, with prior knowledge of the lifetime componentspresent, the potential to multiplex fluorescence lifetimes has beendemonstrated.

19. Co-localisation of Bovine Serum Albumin (BSA) Labelled withDifferent Acridone Dyes Using SDS PAGE 19.1 Preparation of6-(9-oxo-9H-acridin-4-carboxamido)hexanoic acid—bovine serum albumin(BSA) conjugate (Conjugate 3)

To 1 ml of BSA (10.0 mg/ml in 0.1M NaHCO₃ solution) was added a solutionof O—(N-succinimidyl)-6-(9-oxo-9H-acridin-4-carboxamido)hexanoate(lifetime 4 ns) (25 μl; 0.3125 mg/ml in DMSO). The resulting mixture wasincubated at room temperature for 30 minutes with occasional mixing. APD10 column (Amersham Biosciences) was equilibrated with 10 ml ofphosphate buffered saline (PBS; pH 7.4). The dye-labelled BSA was addedto the column, the column washed with PBS (2 ml) and then eluted with 3ml of PBS. The eluate was collected to yield Conjugate 3.

19.2 Preparation of 6-(9-oxo-9H-acridin-10-yl)hexanoic acid—bovine serumalbumin (BSA) conjugate (Conjugate 4)

To 1 ml of BSA (10.0 mg/ml in 0.1 M NaHCO₃) was added a solution ofO—(N-succinimidyl)-6-(9-oxo-9H-acridin-10-yl)hexanoate (lifetime 14 ns)(2 mg in 100 μl of DMSO). The resulting mixture was incubated at roomtemperature for 30 minutes with occasional mixing. A PD10 column(Amersham Biosciences) was equilibrated with 10 ml of phosphate bufferedsaline (PBS; pH 7.4). The dye-labelled BSA was added to the column, thecolumn washed with PBS (2 ml) and then eluted with 3 ml of PBS. Theeluate was collected to yield Conjugate 4.

19.3 Sample Preparation and Electrophoresis

Conjugates 3 and 4 prepared as above, were mixed together in a ratio of2:1 in 0.05M Tris (20 μl; buffered to pH 7.5 with acetic acid containing1% w/v sodium dodecyl sulphate, Bromophenol Blue (10 mg/100 ml) anddithiothrietol (154 mg/100 ml) (Amersham Biosciences). Protein sampleswere reduced by heating to 95° C. for 3 minutes. Electrophoresis wasperformed using a MultiPhor II flat bed electrophoresis system withExcelGel SDS buffer strips (anode strip: 0.45 mol/Tris/acetate pH 6.6, 4g/L SDS and 0.05 g/L Orange G; cathode strip: 0.08 mol/L Tris, 0.80mol/L Tricine and 6.0 g/L SDS pH 7.1). Duplicate samples were applied tothe surface of a pre-formed Excel 8-18 SDS PAGE gradient gel (AmershamBiosciences) using a paper sample application strip placed at locationscorresponding with 96-well microplate centres required for scanning thegel. Molecular weight markers were run in separate lanes, so that partof the gel could be stained using Coomassie Blue to check the integrityof the samples, monitor molecular weight and to orientate the gel forlifetime scanning. Electrophoresis was initiated at constant current toa maximum voltage of 500V for 85 minutes with the flat bed temperaturemaintained at 15° C. Prior to scanning, the gel was fixed in aqueoussolution of 25% methanol, 5% acetic acid v/v for at least 30 minutes.Following lifetime scanning, the gel was stained for 10-20 minutes in0.1% Coomassie Blue G-250 in aqueous solution of 25% methanol, 5% aceticacid v/v and de-stained in aqueous solution of 25% methanol, 5% aceticacid v/v.

19.4 Scanning

Fixed gels were scanned with single wavelength laser excitation at 405nm and the gel sampled at approximately 2 mm intervals along the axis ofthe electrophoretic separation. Data was analysed using a Bayesianalgorithm to assign fluorescence intensity to fluorescence lifetimes of2 ns (gel background); 4 ns (4 ns acridone dye); 6 ns (intrinsic BSAlifetime); and 14 ns (14 ns acridone dye).

FIG. 6 shows the 4 ns dye-labelled BSA and the 14 ns dye-labelled BSA(mixed in a ratio of 2:1) and co-electrophoresed in the same gel lane.Two peaks are resolved at 4 ns and 14 ns, both corresponding to theposition of BSA relative to molecular weight markers. The two labelledBSA species are co-located but distinguishable by lifetime discriminatedintensity. Both BSA species are resolved by the gel to the same locationin the gel as indicated by reference to molecular weight markers andpost electrophoresis staining (66 kD).

The above mentioned examples of conceivable embodiments are intended toillustrate the present invention and are not intended to limit the scopeof protection claimed by the following claims.

1-36. (canceled)
 37. A method for the assay of an analyte in a sample,the method comprising: i) contacting the analyte with a specific bindingpartner for said analyte under conditions suitable to cause the bindingof at least a portion of said analyte to said specific binding partnerto form a complex and wherein one of said analyte and said specificbinding partner is labelled with a fluorescent dye of formula:

wherein: groups R² and R³ are attached to the Z¹ ring structure andgroups R⁴ and R⁵ are attached to the Z² ring structure; Z¹ and Z²independently represent the atoms necessary to complete one ring, twofused ring, or three fused ring aromatic or heteroaromatic systems, eachring having five or six atoms selected from carbon atoms and optionallyno more than two atoms selected from oxygen, nitrogen and sulphur; atleast one of groups R¹, R², R³, R⁴ and R⁵ is the group -E-F where E is aspacer group having a chain from 1-60 atoms selected from the groupconsisting of carbon, nitrogen, oxygen, sulphur and phosphorus atoms andF is a target bonding group; when any of said groups R², R³, R⁴ and R⁵is not said group -E-F, said remaining groups R², R³, R⁴ and R⁵ areindependently selected from hydrogen, halogen, amide, hydroxyl, cyano,amino, mono- or di-C₁-C₄ alkyl-substituted amino, sulphydryl, carbonyl,carboxyl, C₁-C₆ alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl,C₁-C₂₀ alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammoniumand the group —(CH₂—)_(n)Y; and, when group R¹ is not said group -E-F,it is selected from hydrogen, C₁-C₂₀ alkyl, aralkyl and the group—(CH₂—)_(n)Y; Y is selected from sulphonate, sulphate, phosphonate,phosphate, quaternary ammonium and carboxyl; and n is an integer from 1to 6; ii) measuring the fluorescence lifetime of the labelled complex;and iii) correlating the fluorescence lifetime with the presence or theamount of said analyte in said sample.
 38. The method of claim 37,wherein said fluorescent dye has a fluorescence lifetime in the rangefrom 2 to 30 nanoseconds.
 39. The method of claim 37, wherein Z¹ and Z²independently represent the atoms necessary to complete a phenyl or anaphthyl ring structure.
 40. The method of claim 37, wherein said targetbonding group F comprises either: i) a reactive group selected fromcarboxyl, succinimidyl ester, sulpho-succinimidyl ester, isothiocyanate,maleimide, haloacetamide, acid halide, hydrazide, vinylsulphone,dichlorotriazine and phosphoramidite; or ii) a functional group selectedfrom hydroxy, amino, sulphydryl, imidazole, carbonyl including aldehydeand ketone, phosphate and thiophosphate.
 41. The method of claim 37,wherein said spacer group E is selected from: —(CHR′)_(p)——{(CHR′)_(q)—O—(CHR′)_(r)}_(s)— —{(CHR′)_(q)—NR′—(CHR′)_(r)}_(s)——{(CHR′)_(q)—(CH═CH)—(CHR′)_(r)}_(s)— —{(CHR′)_(q)—Ar—(CHR′)_(r)}_(s)——{(CHR′)_(q)—CO—NR′—(CHR′)_(r)}_(s)——{(CHR′)_(q)—CO—Ar—NR′—(CHR′)_(r)}_(s)— where R′ is hydrogen, C₁-C₄alkyl or aryl, which may be optionally substituted with sulphonate, Aris phenylene, optionally substituted with sulphonate, p is 1-20,preferably 1-10, q is 0-10, r is 1-10 and s is 1-5.
 42. The method ofclaim 37, wherein at least one of groups R¹, R², R³, R⁴ and R⁵ comprisesthe group —(CH₂—)_(n)Y, where Y is selected from sulphonate, sulphate,phosphonate, phosphate, quaternary ammonium and carboxyl and n is zeroor an integer from 1 to
 6. 43. The method of claim 37, wherein saidanalyte-specific binding partner pairs are selected from the groupconsisting of antibodies/antigens, lectins/glycoproteins,biotin/streptavidin, hormone/receptor, enzyme/substrate or co-factor,DNA/DNA, DNA/RNA and DNA/binding protein.